LC-MS in Analytical Toxicology

Special Issue: Review Article
Received 30 September 2010, Accepted 4 October 2010 Published online in Wiley Online Library: 10 December 2010
(wileyonlinelibrary.com) DOI 10.1002/bmc.1566
LC-MS in analytical toxicology: some practical considerations

Lewis Couchman and Phillip E. Morgan*
ABSTRACT: Liquid chromatography, coupled with single-stage or tandem mass spectrometry, is a powerful tool increasingly used in analytical toxicology. However, the atmospheric pressure ionization processes involved are complex, and subject to interference from matrix components, for example. Further, the techniques used in sample preparation, chromatography and mass analysis are developing rapidly. An understanding of the advantages and limitations of LC-MS ensures appropriate analyses are performed, and that reliable results are generated. Consideration should be given to the inuence of the sample preparation and chromatographic conditions on the ionization of the analyte at the mass spectrometer interface. This review aims to provide some practical guidance and examples to aid method development for commonly encountered analytes in analytical toxicology. Copyright 2010 John Wiley & Sons, Ltd. Keywords: liquid chromatography; mass spectrometry; analytical toxicology; sample preparation
Introduction
Analytical toxicology is the detection, identication and measurement of drugs and other foreign compounds (xenobiotics) and their metabolites in biological and related specimens. Analyses tend to fall into (i) emergency and general hospital toxicology, including poisons screening or (ii) more specialized categories such as forensic toxicology, screening for drugs of abuse, therapeutic drug monitoring (TDM) and occupational/ environmental toxicology. However, there is considerable overlap between all of these areas. Sample matrices can be complex, particularly in the case of post-mortem analyses, and a high degree of analytical reliability, sensitivity and specicity may be required (Maurer, 2006, 2007; Flanagan et al., 2007). Since the rst report of an interface between liquid chromatography and mass spectrometry a number of interface designs, most importantly that of atmospheric pressure ionization (API), have been developed to improve the eciency of the ionization process. A better understanding of the physical processes involved with analyte ionization means that problems associated with co-eluting matrix components (ion suppression and enhancement) can be accounted for and minimized. Whilst gas chromatographymass spectrometry (GC-MS)in conjunction with detailed GC-MS spectral librariesremains a very useful tool for systematic toxicological analysis (STA), non-volatile, polar (e.g. conjugated metabolites), and thermally labile compounds are dicult or impossible to analyse without lengthy derivatization procedures (Marquet and Lachtre, 1999; Flanagan et al., 2007; Dresen et al., 2010). HPLC with diode-array detection (DAD) provides a means to analyse compounds not suited to GC, but suers due to the non-specic nature of UV detection. Certain compounds of toxicological relevance also have poor UV absorbance. LC-MS (and LC-tandem MS, LC-MS/MS) may be applied to compounds not suited to GC analysis, and spectral libraries now exist for a very wide range of toxicologically relevant compounds (although ionization and fragmentation conditions remain nonstandardized). Recent developments in accurate mass measure-
ment have allowed tentative identication of compounds without the absolute need for reference materials. An understanding of the advantages and limitations of MS methods may help generate reliable quantitative and qualitative data. Sample collection/pre-treatment procedures and protocols,
* Correspondence to: P. E. Morgan, Toxicology Unit, Department of Clinical Biochemistry, Kings College Hospital NHS Foundation Trust, Denmark Hill, London SE5 9RS, UK. E-mail: phillip.morgan@nhs.net Toxicology Unit, Department of Clinical Biochemistry, Kings College Hospital NHS Foundation Trust, Denmark Hill, London SE5 9RS, UK Abbreviations used: 6-MAM, 6-monoacetylmorphine; AAFS, The American Academy of Forensic Sciences; ACN, acetonitrile; APCI, atmospheric pressure chemical ionization; API, atmospheric pressure ionization; APPI, atmospheric pressure photoionization; BEG, benzoylecgonine; CID, collision-induced dissociation; CNS, central nervous system; DAD, diode-array detection; DMA, dimethoxyamphetamine; DMANO, dimethylamphetamine N-oxide; DoA, drugs of abuse; DVB, divinylbenzene; EDDP, 2-ethylidene-1,5-dimethyl-3,3diphenylpyrrolidine; EDTA, ethylene diamine tetra-acetic acid; EMDP, 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline; ESI, electrospray ionization; GC-MS, gas chromatographymass spectrometry; GHB, gamma hydroxybutyrate; GUS, general unknown screening; H-ESI, heated electrospray ionization; HILIC, hydrophilic interaction liquid chromatography; HRMS, high-resolution mass spectrometry; IPA, isopropyl alcohol/2-propanol; ISTD, internal standard; LC-MS, liquid chromatographymass spectrometry; LC-MS/MS, liquid chromatographytandem mass spectrometry; LLE, liquid liquid extraction; LLoQ, lower limit of quantitation; LOD, limit of detection; M3G, morphine-3-glucuronide; M6G, morphine-6-glucuronide; MBDB, methylbenzodioxolylbutanamine; MDA, 3,4-methylenedixoyamphetamine; MDEA/MDE, 3,4-methylenedioxyethylamphetamine; MDMA, 3,4methylenedioxymetamphetamine; MEPS, micro-extraction by packed sorbent; MTBE, methyl tert-butyl ether; PCP, phencyclidine; PPT, protein precipitation; RAM, restricted access material; RP, reversed-phase; SCX, strong cation-exchange; SOFT, The Society of Forensic Toxicologists; SPE, solidphase extraction; SRM, selected reaction monitoring; STA, systematic toxicological analysis; TDM, therapeutic drug monitoring; TFA, triuoroacetic acid; THC, tetrahydrocannabinol; THC-COOH, carboxytetrahydrocannabinol; TOF, time of ight; TRIS, tris(hydroxymethyl)aminomethane; UHPLC, ultra-high-pressure liquid chromatography.
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LC-MS in analytical toxicology and choice of sample preparation and HPLC conditions all inuence the nal result (Flanagan et al., 2005, 2007; Dinis-Oliveira et al., 2010). This review highlights some practical points for consideration when using LC-MS (or MS/MS) for analysis of the most common biological samples encountered in analytical toxicology laboratories. for analytes possessing ionizable groups often adds selectivity to the procedure, as well as helping to optimize recovery of analyte(s) from the matrix and ensuring reproducible sample consistency (Hendriks et al., 2007). Changes in sample pH may aect the recovery of other analytes, therefore conditions are often a compromise, particularly when multiple analytes from dierent classes are to be simultaneously investigated. Control of pH is often through the use of buer solutions such as tris(hydroxymethyl)-aminomethane (TRIS, pH range 79), sodium acetateacetic acid solutions (pH range 3.56), citric acidcitrate solutions (pH range 36) and carbonatebicarbonate solutions (pH range 911). In our experience, solutions of TRIS (2 mol/L) can be used to good eect even at pH 10.6 for the extraction of basic drugs from serum or plasma (Flanagan et al., 2001; Morgan et al., 2003). Detailed lists of compounds and their properties are available, for example http://www.sigmaaldrich.com/life-science/core-bioreagents/ biological-buers/learning-center/buer-reference-center.html (accessed 10 August 2010). If pH control is less of an issue during sample preparation, simple acidication or alkalinization may be achieved by addition of strongly acidic or alkaline aqueous solutions. However, some analytes decompose, undergo structural rearrangements or react under these conditions, leading to erroneous results. This is particularly true for certain metabolites, such as N-oxides, but it can also be useful, for example, in the conversion of glucuronidated metabolites to the parent compound. Special care must be taken not to introduce non-volatile buer salts into the mass spectrometer. Other considerations are listed in Table 1. The most common sample preparation techniques currently employed in analytical toxicology are protein precipitation (PPT), liquidliquid extraction (LLE), and solid-phase extraction (SPE). In the following sections, selected applications will be used in order to highlight the dierent approaches taken. Protein Precipitation The precipitation of proteins from biological uids is rapid and simple, and the eciency of various precipitation reagents has been evaluated (Blanchard, 1981). Chambers et al. (2007) showed that, for the compounds testeda range of eight representative polar and non-polar analytesrecovery was generally good (76 114%) following PPT of plasma. In particular, recovery of polar analytes included in the test was better than the three LLE methods used for comparison, and comparable to two of the three SPE methods investigated. However, matrix eects were
Sample Preparation
Sample preparation prior to LC-MS analysis aims to reduce matrix eects via removal of potential interferences, and to get the analyte into a form amenable to analysis. However, for drugs and low molecular mass compounds, co-eluting components such as proteins, lipids and salts may cause variability in the eciency of analyte ionization (Bonglio et al., 1999; Jemal et al., 2010). Nonvolatile components may also cause a reduction in sensitivity and form deposits inside the instrument. The removal of phospholipids from plasma/whole blood, compounds known to cause signicant ion suppression in many cases, is the basis for a number of reports comparing the eciency of certain sample preparation techniques (Little et al., 2006; Chambers et al., 2007; Ismaeil et al., 2008; Du and White, 2008; Pucci et al., 2009). As well as sample clean-up, analytes can also be concentrated or diluted during sample preparation, depending on factors such as sample volume and the anticipated analyte concentration(s). Poor performance may result if sample preparation is overlooked (Van Eeckhaut et al., 2009). That said, the superior selectivity of MS detectors coupled with the versatility of HPLC has prompted the simplication, miniaturization and greater automation of sample preparation processes. For STA, the direct injection of urine samples, usually after ltration or centrifugation and/or dilution (dilute and shoot) has been shown to be useful. Advantages include increased selectivity and lower limits of detection (LODs) in many cases. However, the direct injection strategy is prone to signicant variations in matrix eects. The general techniques for the preparation of solid, liquid and gaseous samples for chromatographic analyses have been reviewed (Smith, 2003; Flanagan et al., 2006; Chen et al., 2008; Novkov and Vlov, 2009). General Considerations The physicochemical properties of the analyte(s), for example pKa, and octanolwater coecient (as logP) can help guide towards an appropriate sample preparation procedure (Flanagan et al., 2007). In liquid samples, for example, manipulation of pH
Table 1. Some sample preparation considerations Minimize matrix eects as far as practical by (i) removal of endogenous interferences, e.g. phospholipids and (ii) the use of appropriate internal standard(s). Is the chosen method cost eective? Consider the time spent preparing samples, the number of steps involved, and the cost of reagents and materials. Is it possible to automate the procedure for high-thoughput analyses? Does the method give suitable analyte recovery? Recovery should be reproducible, and independent of analyte concentration. Evaporation steps should not degrade the analyte(s). The use of an inert gas (e.g. nitrogen) and temperatures as low as practical are recommended. Additional measures, such as acidication of the eluate prior to evaporation, may be required to minimize loss of amphetamine and related compounds (Mortier et al., 2002). Logistical considerations, e.g. fume hood(s), provision of vacuum and compressed air and/or nitrogen, bench space required. Environmental/health and safety impact.
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L. Couchman and P. E. Morgan considerable, ranging from 47 to 61% suppression of ionization. Moreover, the choice of methanol or acetonitrile as the precipitating reagent also aected the abundance of residual phospholipids in the supernatant. Although the acetonitrile-treated samples contained substantially less phospholipids, the interference was such that a fully validated method was not deemed viable. However, validated methods have been published (Table 2). Evaporation of the supernatant and reconstitution of the residue in mobile phase gave good results in the analysis of lamotrigine and metabolites in plasma (Beck et al., 2006), with no apparent ion-suppression. Dilution of the supernatant prior to injection is also an option, and can be adjusted according to the expected analyte concentration. Using this approach, Kirchherr and Khn-Velten (2006) reported negligible matrix eects for all analytes with the exception of olanzapine, for which the use of matrix-matched calibration solutions was mandatory. The poor ability of PPT methods to remove certain phospholipids from plasma/serum (Chambers et al., 2007; Jemal et al., 2010) means that further treatment of the supernatant, such as SPE or LLE, may be necessary to reduce ion-suppression and to enhance sensitivity (Flanagan et al., 2006). Chilling the precipitation reagent prior to use may increase recovery of analytes and improve reproducibility (Choo et al., 2007). For whole blood and other dirty sample matrices, a PPT step is often employed sometimes with ultrasonicationbefore the supernatant is subjected to SPE (Kristoersen et al., 2007; Marin et al., 2008; Mercerolle et al., 2008; Chimalakonda et al., 2010; Wu et al., 2010). Protein precipitation is clearly an attractive sample preparation technique due to its speed, simplicity and the good recovery of polar analytes compared with some SPE and LLE procedures. It is applicable to a range of LC-MS methods relevant to toxicology. However, the failure to remove endogenous phospholipids and other potentially interfering compounds make it prone to severe matrix eects in the absence of further treatment of the supernatant. Moreover, it is non-selective and dicult to automate, and there may be considerable variation in the eectiveness of precipitation between samples, even those of the same matrix. LiquidLiquid Extraction Given appropriate conditions, many analytes readily partition into an organic phase from an aqueous sample, the extent of partitioning being based on the octanolwater partition coecients of the analytes. The ideal organic phase is immiscible with the sample matrix, of low toxicity, volatility and ammability, and eciently extracts the analytes of interest without also extracting endogenous material. Many solvents are used (Table 3), none of which meet the ideal criteria listed, and most of which require specialized storage, handling and disposal procedures. Polar organic solvents such as methyl tert-butyl ether (MTBE) and 1-chlorobutane tend to extract fewer interferences such as phospholipids. Depending on the analyte(s), the use of acidic pH conditions further helps to prevent co-extraction of these compounds. Hence, these solvents have been recommended as the best single-component solvents in LLE in terms of analyte recovery and extract cleanliness (Jemal et al., 2010). However, other solvents may be better suited to particular analytes, matrices or conditions, and should not be excluded (Srinivas, 2009). Ethyl acetate LLE gave cleaner extracts from urine compared with SPE for analysis of doping agents (Goebel et al., 2004), but caused problems with the extraction of certain diuretics containing sulfur side chains. This was overcome by using MTBE instead of ethyl acetate, the caveat being poor recovery of the other analytes. Mixtures of various solvents can also prove useful. A rather elaborate extraction solvent (2 mL/ sample) consisting of a mixture of dichloromethane (520 mL), dichloroethane (520 mL), heptane (600 mL) and 2-propanol (380 mL) was used to extract benzodiazepines from urine (Glover and Allen, 2010). Various LLE solvent and buer combinations were evaluated for the extraction of 19 antipsychotic drugs from whole blood (Saar et al., 2009). In that study, TRIS buer at pH 9.2 (1 mL, 2 mol/L) gave the best extraction eciency regardless of extracting solvent used, as compared with sodium sulfate (1 mL, saturated) and sodium bicarbonate (100 mg). Of the solvents used (8 mL of 1-chlorobutane, ethyl acetate or a 50:50 diethyl etherethyl acetate mixture), 1-chlorobutane gave the highest extraction eciency for all analytes, with the exception of sulpiride. Matrix eects were broadly similar between the tested solvents; however, increased matrix eects were observed for olanzapine when extracted using ethyl acetate. In an evaluation of several extraction methods for methadone in human plasma, LLE using hexane isoamyl alcohol was more ecient and less likely to cause ion suppression than mixed mode SPE, protein precipitation, or column switching arrangements (Souverain et al., 2004). A mixture of butyl acetate : butanol (9 + 1, v/v) was used for the extraction of amphetamine, metamphetamine and amisulpride from serum/plasma (Couchman et al., 2010a), and is applicable to other antipsychotic drugs and amphetamine-related compounds. MTBE does not extract phospholipids (Jemal et al., 2010), but can give poor recovery of polar compounds (Chambers et al., 2007). The use of basied solvent and multiple extractions of the same sample can help improve sensitivity, although the latter is somewhat tedious. MTBE was preferred over toluene and butyl acetate for extracting antipsychotics from post-mortem blood (Roman et al., 2008). A general assumption is that for selective LLE of ionizable analytes, the sample pH should be adjusted to a value at least 2 units above or below the analyte pKa for basic or acidic analytes, respectively. However, in certain circumstances (and indeed in our own experience), enhanced selectivity and good recoveries can be achieved whilst the analyte is apparently largely ionized (Hendriks et al., 2007). Liquidliquid extraction is simple, robust and transferable, and shows good reductions in matrix eects (Guo et al., 2005; Jemal et al., 2010). It may be more suited to urgent analyses than SPE (Flanagan et al., 2006; Wille and Lambert, 2007). However, it may not be suitable for hydrophilic compounds, and the formation of emulsions can make it dicult to isolate the extraction solvent. Appropriate treatment of the sample prior to addition of the solvent can give highly selective extractions in some cases, and direct injection of the extract may also be possible. In most cases, though, there is a need to evaporate the solvent extract before re-dissolving the residue in mobile phase. This extra step costs time and increases error. Storage, handling of relatively large volumes, toxicity, disposal and the cost of and need for high-purity solvents used in LLE are also issues for consideration. Solid-phase Extraction SPE involves application of the sample onto a bed of material akin to the stationary phase in HPLC. Chemical modication of the
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Table 2. Selected direct injection/protein precipitation methods in analytical toxicology LC-MS Matrix Urine Urine Urine Urine For screening: 10 mL For conrmation: 1 mL Screen: diluted before injection Conrm: SPE (C18) 500 mL/10 mL 20 mL/10 mL 100 mL/5 mL Diluted before injection Diluted before injection Diluted before injection Sample/injection volume Comments Reference Badoud et al., 2009 Gustavsson et al., 2007 Thrngren et al., 2008 Nordgren and Beck, 2004
LC-MS in analytical toxicology
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Analyte(s)
Urine Urine Urine Plasma 1 mL/5 mL 1 mL/100 mL 100 mL/10 mL
20 mL/10 mL Diluted before injection. LOD 243 ng/mL Centrifuged before injection
Andersson et al., 2008 Dams et al., 2003a Edinboro et al., 2005 Umezawa et al., 2008
Doping agents (103 analytes) Opiates (8 analytes) Diuretics, CNS stimulants, opiates (130 analytes) Phenylethylamines, N-benzylpiperazine, non-benzodiazepine hypnotics (23 analytes) Amphetamine, metamphetamine, MDMA, MDA Opioids, cocaine, and metabolites (25 analytes) Opiates (10 analytes) b-Blockersacebutolol, labetalol, metoprolol, propranolol Plasma and urine 1 mL/180 mL
Etizolam, brotizolam, lorazepam
Lee et al., 2003
Oral uid
500 mL/50 mL
Centrifuged before injection Diluted before injection onto size exclusion HPLC column. LOD 13 ng/mL Diluted and ltered before injection onto size exclusion HPLC column. LOD 25 ng/mL Diluted before injection. In-house HPLC column packing required due to limited column lifetime (200 injections)
Allen et al., 2005
Morphine, 6-MAM, codeine, dihydrocodeine, methadone, EDDP, cocaine, benzoylecgonine, diazepam, nitrazepam, nordiazepam, temazepam, 7-aminonitrazepam Bupropion, hydroxybupropion, threohydrobupropion Post-mortem blood and urine Oral uid 90 mL 100 mL
Mercerolle et al., 2008
Amphetamine, metamphetamine, MDA, MDE, MDMA, cocaine, benzoylecgonine, ketamine, phencyclidine, psylocibine, mescaline
Blood: PPT (methanol : ZnSO4), then mixed mode SPE Urine: mixed mode SPE. LOD 5 mg/L PPT (methanol), sonication (6 min) then m-SPE. LOD 0.051.2 ng/mL
Sergi et al., 2010
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Matrix Blood and urine 500 mL Sample/injection volume Comments Reference Wu et al., 2010 Serum 100 mL Bouzas et al., 2009 Breast milk 500 mL Choo et al., 2007 Post-mortem blood Oral uid and hair Oral uid: 200 mL 1 mL BloodPPT (ACN), then mixed mode SPE. Urineacidied then mixed mode SPE. LOD 0.1 to 1 ng/mL in urine, 0.11.5 ng/mL in blood PPT (MeOH : ZnSO4), evaporate and reconstitute then online column switching SPE. LOD < 1 ng/mL PPT (chilled methanol), then mixed mode SPE. LOD 5 ng/mL methadone and EDDP, 10 ng/mL EMDP PPT (acetone) Herrin et al., 2005 Doherty et al., 2007 Serum 100 mL Kirchherr and Khn-Velten, 2006 Edwards and Smith, 2005 PPT (MeOH). Sonication (2 min plasma, 6 min oral uid), then ltered. LOD 0.22.8 ng/mL (plasma), 13.7 ng/mL (oral uid) Sergi et al., 2009 Serum (rat) Plasma and oral uid 150 mL 50 mL Oral uid: PPT, evaporate & reconstitute. Hair: soxhlet extraction PPT (MeOH : ACN, 1 + 9). Supernatant diluted before injection PPT (ACN) Serum 500 mL Ming and Heathcote, 2009 L. Couchman and P. E. Morgan Plasma 200 mL PPT (ACN). Supernatant diluted before injection. LOD 15 pg/mL clozapine, 10 pg/mL norclozapine PPT (ACN). LOD lamotrigine 0.08 mmol/L Beck et al., 2006
Table 2. Continued
Analyte(s)
Alkaloidsaconitine, hypaconitine, gelsemine, raceanisodamine, strychnine, brucine. High percentage organic (957545%) for better ionization/sensitivity Basic drugs of abuse (18 analytes)
Methadone, EDDP, EMDP
Various drugs (114 analytes). Screen and identify Risperidone, sertraline, paroxetine, trimipramine, mirtazapine, plus nine metabolites (14 analytes) Antidepressants and antipsychotics (48 analytes)
Morphine, M3G, oxycodone, noroxycodone Amphetamine, metamphetamine, morphine, 6-MAM, MDA, MDE, MDMA, cocaine, BEG, THC, THC-COOH, ketamine, phencyclidine Clozapine and norclozapine
Lamotrigine + 3 metabolites
Table 3. Selected liquidliquid extraction methods in analytical toxicology LC-MS Matrix Urine Urine 3 mL 2 mL Sample requirements Extraction solvent(s) Reference Mazzarino et al., 2010 Deventer et al., 2002
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Analyte(s)
Tamoxifene metabolites (6 analytes) Diuretics and probenecid (18 analytes) Blood, urine, hair 250 mL (blood/urine) 20 mg (hair) 1 mL 1g
Ethyl acetate or MTBE Ethyl acetate. Double extraction (2 portions of solvent). LOD 2100 ng/mL 1-Chlorobutane. Large volumes of solvent
Laloup et al., 2005
Urine Post-mortem blood
ElSohly et al., 2006 Roman et al., 2008
Chloroform : isopropyl alcohol (9 + 1). LOD 0.53 ng/mL MTBE. LOD better than 1 mg/L
Plasma Plasma 500 mL
1 mL
Moody et al., 2002 Remane et al., 2010
1-chlorobutane : acetonitrile (4 + 1). LLOQ 0.1 ng/mL Butyl acetate : ethyl acetate (1 + 1)
Urine
5 mL
Diethyl ether. LOD 110 ng/mL
Deventer et al., 2006
Benzodiazepines and metabolites, zolpidem and zopiclone (28 analytes) Benzodizepines (22 analytes). LC-MS-(ToF) Risperidone, 9-OH risperidone, busiprone, ziprasidone, perphenazine, zuclopenthixol, uphenazine, upenthixol Buprenorphine, norbuprenorphine, naloxone Various classes: antidepressants, benzodiazepines, neuroleptics, beta-blockers, oral antidiabetics, brain-death diagnoses analytes (140 analytes) Anabolic steroids (tetrahydrogestrinone, gestrinone, 3-hydroxystanozolol, 17a-trenbolone) Amphetamine, metamphetamine, MDA, MDMA, PCP Benzodiazepines (17 analytes) Oral uid Urine 1 mL 500 mL
Kala et al., 2008 Glover and Allen, 2010
Hexane : ethyl acetate (1 + 1). LOD 210 ng/mL Dichloromethane : dichloroethane : heptane : 2-propanol (52 + 52 + 60 + 38). LOD 0.312.5 mg/L
L. Couchman and P. E. Morgan packed bed to provide similar chemistries to the packings used in HPLC means that a large number of dierent phases are available, in many formats, making SPE a highly versatile technique. For many SPE materials, allowing the sorbent to become dry risks inactivation of the bonded phase, leading to poor recovery of analytes, and to channelling of the sample through the packed bed. However, some newer, polymeric sorbents are more resistant to this kind of problem, can tolerate relatively dirty samples and are stable over a wider pH range compared with silica-based materials (Yawney et al., 2002). Polymeric sorbents may also be less soluble in certain solvents compared with silica-based material (Verplaetse and Tytgat, 2010). The presence of some particulates is generally tolerated, but the beds can become blocked. To overcome this, samples can be ltered or centrifuged, after any pH adjustment. Once eluted from the cartridge, the eluate is most often evaporated and reconstituted. A mobile phase containing a high proportion of organic solvent may facilitate the direct injection of organic sample extracts, saving time and reducing the risk of errors (Couchman et al., 2010a), but is obviously dependant on the initial chromatographic conditions chosen. Direct injection, and dilution of the eluate prior to injection have been reported (Table 4). Generic SPE methods have been proposed, not necessarily intended for the analysis of every analyte, but to provide a set of starting conditions with a reasonable chance of success with minimal adjustment. Schellen et al. (2003) evaluated several SPE materials for extraction of a range of drugs from serum/plasma, noting that a divinylbenzene (DVB) material oered the best combination of extraction capacity and desorption eciency amongst those tested. A series of SPE sorbents ranging from non-polar, to mixed-mode, to polymeric, were tested for their performance in the systematic toxicological analysis of a diverse range of drugs (17 analytes) in whole blood (Decaestecker et al., 2003). In this experiment, a C8-modied silica material was found to oer the best overall recovery, followed closely by a mixed-mode polymeric sorbent. It was reported that the C8 material oered the cleanest sample extracts. Dierences in analyte ionization eciencies and concentrations in the sample may be compensated for by adjustment of sample volumes and/or the sorbent mass used for SPE (Rentsch, 2003; Maralikova and Weinmann, 2004). Silica-based C8 and C18 sorbents generally oer predictable chemistry and good recovery of a wide range of analytes from various matrices, often without the use of extreme pH values. However, they are relatively nonselective, and good recoveries of polar, hydrophilic, and ionized compounds may be dicult. So-called mixed-mode materials, in which a combination of interactions may be exploited to allow ecient clean-up by using relatively harsh wash steps with minimal loss of analyte(s), are increasingly used for sample preparation. Such methods typically involve the sequential elution of acidic, neutral and basic compounds using solvents at appropriate pH, and this versatility has led to their increasing popularity. For comprehensive analyses, the eluates from dierent fractions are usually combined and evaporated before reconstitution in an LC-compatible solvent. Otherwise only the fraction containing the analytes of interest is collected. Control and manipulation of pH is often the key in these cases. A diverse range of applications in which mixed-mode SPE has been used have been reported (Table 4). Advantages of SPE include the extraction of relatively hydrophilic compounds such as metabolites of morphine and cocaine (Yawney et al., 2002; Jagerdeo et al., 2008), enhanced selectivity imparted through chemical modication of the particle surface, ease of automation, high sensitivity and high eciency. Compared with LLE, SPE is considered to use less solvent, is less time-consuming, and gives cleaner extracts, especially from ante-mortem blood, plasma or serum (Chambers et al., 2007). The use of 96-well SPE plates can increase sample throughput, whilst at the same time reducing sample and solvent volumes (Mallet et al., 2003; Ashman et al., 2010). Limitations of SPE include co-extraction of interfering compounds, and poor extraction of some drugs (Yawney et al., 2002; Goebel et al., 2004). The latter was addressed to some extent by Schellen et al. (2003) in using a larger sorbent bed for the extraction of a very polar compound (acetaminophen), and by acidication of the sample (20 mL phosphoric acid/mL of sample) to improve recovery of sulfadiazine and sulfamerazine. Another problem may be blockage of the packed bed during sample application. The use of larger SPE cartridges, and/or centrifugation, ultrasonication, protein precipitation or dilution of the sample can be useful in these cases, especially when viscous samples are encountered (Choo et al., 2007; Saar et al., 2009). The sensitivity of modern instruments is such that the dilution of extracts may be routinely required in order to maintain concentrations within the linear range of the mass spectrometer (Langman et al., 2009). Batch-to-batch reproducibility of the sorbent bed, although less of a problem now compared with a few years ago, is still a concern (Novkov and Vlov, 2009) and without automation the number of samples that can be processed simultaneously is generally limited to the number of spaces available on the vacuum manifold, typically 20. Flow-rate through the sorbent bed is dicult to control in most cases, which can lead to variable analyte recovery. Although the 96-well plate SPE format has advantageslow volumes of sample and solvents, small desorption volumes, decreased void volumes, semi-automationthere are also disadvantages particularly in terms of cost, when not all the wells are used each time. Micropipette tip-based SPE may oer an alternative (Shen et al., 2006). As well as selectivity, trace enrichment and ease of use compared with LLE and PPT, procedures involving SPE are relatively easy to automate (Yawney et al., 2002). This can save time and reduce labour costs, and at the same time enhance forensic integrity (Jagerdeo et al., 2008; Robandt et al., 2009). Instruments for semi-automated or fully automated SPE have been available for some years. Despite applications demonstrating robustness, reliability and time-savings (Jourdil et al., 2003; Schellen et al., 2003; Goebel et al., 2004; Robandt et al., 2008, 2009), the capital cost of the instrumentation required even for a semi-automated system is likely to limit widespread implementation. Moreover, pre-extraction steps such as addition of internal standard, hydrolysis, pre-mixing of the sample with buer solutions and protein precipitation, and post-extraction evaporation and reconstitution of the eluate often need to be performed o-line (Jourdil et al., 2003; Maralikova and Weinmann, 2004; Kristoersen et al., 2007; Robandt et al., 2009). Other Matrices/Preparation Techniques Direct injection of samples onto size-exclusion HPLC columns have reported for the analysis of benzodiazepines in diluted urine and plasma (Lee et al., 2003, 2006), and for the measurement of b-blockers in diluted plasma (Umezawa et al., 2008). Sample extraction and the need for column switching were eliminated, and good recovery, precision and LODs were reported. Despite
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Table 4. Selected solid-phase extraction methods in analytical toxicology LC-MS Matrix Whole blood Urine Urine Vitreous humor 1 mL 500 mL 1 mL Mixed mode 96 well plate. Direct injection of eluate Mixed mode. Acidic and basic eluate fractions combined Mixed mode. As Pelander et al., 2003. Little sample left if re-analysis required Mixed mode. LOD 530 ng/mL 1 mL Weak cation-exchange sorbent. LOD 3.620.4 ng/mL Sample volume SPE mode and comments Reference Arin and Anderson, 2006 Badoud et al., 2010 Pelander et al., 2003 Pelander et al., 2010
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Analyte(s)
Quaternary ammonium drugs and herbicides (11 analytes) Doping agents (103 analytes) Comprehensive screening (392 <real> analytes, plus 245 <theoretical mass> in library) Comprehensive screening (815 exact masses for DoA, therapeutic drugs, designer drugs. Retention data for half of these) Morphine, codeine, ethylmorphine glucuronides, 6-acetylmorphine Ketamine and selected metabolites (3 analytes) Urine Urine Blood, plasma, urine Urine Oral uid Post-mortem blood and urine Urine Urine 500 mL 500 mL 250 mL 100 mL 2.5 mL 1 mL 4 mL 50 mL Mixed mode. LOD 0.03 (ketamine) and 0.05 (norketamine) ng/mL C18 sorbent. LOD range 0.24.0 ng/mL
Svensson et al., 2007 Parkin et al., 2008
Cannabinoids, opioids and stimulants (13 analytes)
MDMA and metabolites (3 analytes)
Maralikova and Weinmann, 2004 Jenkins et al., 2004 Coulter et al., 2010 Mercerolle et al., 2008 Feng et al., 2007 Fernandez et al., 2007
Mixed mode. Direct injection of eluate. LOD 0.0150.04 mg/mL Mixed mode. LOQ 5 ng/mL Mixed mode. LOD 5 mg/L Mixed mode. All LODs < 3 ng/mL Mixed mode. LOD range 0.00032.5 ng/mL
Urine Urine Plasma
1 mL 1 mL 50 mL
Langman et al., 2009 Robandt et al., 2008 Ashman et al., 2010
Antidepressants (16 analytes) Bupropion, hydroxybupropion, threohydrobupropion Multiple DoA and metabolites (30 analytes) Multiple hallucinogens, chlorpheniramine, ketamine, ritalinic acid, and metabolites (14 analytes) Cocaine and metabolites (7 analytes) Benzoyecgonine, m-hydroxybenzoylecgonine, p-hydroxybenzoylecgonine, norbenzoyecgonine Midazolam, hydroxymidazolam, hydroxymidazolam glucuronide, morphine, morphine-3-glucuronide, morphine-6-glucuronide Amphetamine, metamphetamine, MDA, MDMA, MDEA, ephedrine, pseudoephedrine, phentermine, phenylethylamine Blood 1 mL
Mixed mode. LOD 0.25 ng/mL all analytes C8 sorbent. Direct injection of eluate. LOD 1.2 ng/mL Mixed mode 96-well format. Two separate methods required. Direct injection of eluate. LLOQ < 10 ng/mL Mixed mode
Apollonio et al., 2006
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Matrix Blood 200 mL Sample volume SPE mode and comments Reference Concheiro et al., 2006 Plasma and urine 1 mL Jourdil et al., 2003 Plasma 100 mL Schellen et al., 2003 Urine Serum Plasma 100 mL 100 mL 1 mL Mixed mode. LOD: 0.5 ng/mL for metamphetamine, MDMA, benzoyecgonine, cocaine; 1 ng/mL for morphine, 6-MAM, MDA, MDEA, MBDB Mixed mode. LOD (urine): 0.025 ng/mL unitrazepam and 7-aminounitrazepam; 0.04 ng/mL N-desmethylunitrazepam; 0.2 ng/mL 3-hydroxyunitrazepam Divinylbenzene sorbent. Direct injection of eluate. LLOQ < 1 ng/mL except acetaminophen (2 ng/mL) Mixed mode. LOD < 2 ng/mL Mixed mode. LOD range 0.311.4 ng/mL LOD < 1 ng/mL except for carbamazepine-10,11-diol (9.77 ng/mL) Kim et al., 2008 Nakamura et al., 2009 Subramanian et al., 2008 Blood and urine 500 mL Mixed mode. LOD 0.11 ng/mL in urine; 0.11.5 ng/mL in blood Wu et al., 2010 Urine Serum 1 mL 100 mL Mixed mode. Direct injection of eluate PPT (MeOH : ZnSO4), evaporate and reconstitute then online column switching SPE. LOD < 1 ng/mL Jagerdeo et al., 2008 L. Couchman and P. E. Morgan Bouzas et al., 2009
Table 4. Continued
Analyte(s)
Morphine, 6-acetylmorphine, amphetamine, metamphetamine, MDA, MDMA, MDEA, MBDB, benzoylecgonine, cocaine Flunitrazepam, 7-aminounitrazepam, 3-hydroxyunitrazepam, N-desmethylunitrazepam
Various drugs (acidic, basic, polar, non-polar, aromatic; 11 analytes)
Amphetamine, metamphetamine, MDA, MDMA, MDEA, DMA, DMANO Benzodiazepines (16) and metabolites (5) Zonasimide, lamotrigine, topiramate, phenobarbital, phenytoin, carbamazepine, carbamazepine10,11-diol, 10-hydroxycarbamazepine, carbamazepine-10,11-epoxide Alkaloidsaconitine, hypaconitine, gelsemine, raceanisodamine, strychnine, brucine. High percentage organic (957545%) for better ionization/sensitivity Cocaine, benzoylecgonine, ecgonine methyl ester, ecgonine, cocaethylene Basic drugs of abuse (18 analytes)
LC-MS in analytical toxicology matrix eects of 1130% suppression for the b-blockers, excessive variability was not observed. In a comparison of various LLE and SPE procedures for the extraction of antipsychotics from dierent blood matrices, i.e. ante-mortem, non-decomposed post-mortem, and decomposed post-mortem blood, considerable variation was observed in terms of both analyte recovery and matrix eects (Saar et al., 2009). However, the variability appeared to be related to the matrix rather than to the method of extraction. Dierences have also been seen in the recovery of PCP from serum and from whole blood (Chimalakonda et al., 2010) after extraction using mixed-mode SPE, and attempts to improve the LOD by reducing the volume of solvent used in the nal reconstitution step failed, possibly due to incomplete dissolution of the analyte, and/or concentration of ion-suppressing matrix components. Oral uid is an attractive alternative to urine and plasma, however variations in the matrix warrant stringent method validation. Recovery of analytes from oral uid is often superior when compared with plasma (Sergi et al., 2009). Direct injection of diluted oral uid has been reported, however this resulted in shortened HPLC column lifetimes (Allen et al., 2005). SPE and LLE procedures analogous to those used for urine and plasma samples are usually employed. However, the use of sampling devices can lead to problems with interferences and other contaminants (Mortier et al., 2002; Allen et al., 2005), even after an extraction procedure. The use of a micro-SPE (m-SPE) procedure (Sergi et al., 2010) reduced matrix eects compared with PPT, avoiding the need for a clean-up gradient after each injection. A review of analytical procedures for the analysis of oral uids for drugs of abuse is available (Samyn et al., 2007). Discussion Advances in LC and MS technologies, plus economic pressures, mean that sample preparation centres less on the selective and often lengthy extraction of specic analytes, and more on the removaleither during sample preparation or during chromatographic analysisof species likely to interfere in analysis. Sorbents for SPE have been developed to specically remove phospholipids and proteins from biological samples. Monolithic sorbents less prone to blockages are available as disposable tips or in 96-well plates. Despite the superior results obtained from SPE compared with PPT, the cost in terms of labour and materials may still be dicult to justify (Mallet et al., 2003). Automated extraction is routine within the pharmaceutical industry, but limited in the toxicology laboratory at present. Turbulent ow chromatography oers ecient removal of potential interferences (Du and White, 2008), and fast analyses from biological uids when compared with SPE or LLE procedures (Berna et al., 2004; Zhou et al., 2005; Morgan et al., 2010). Oine handing of the sample is often limited to centrifugation and dilution (Couchman et al., 2010b). Moreover, in the same way that automated SPE systems can be congured for minimum cycle times (Schellen et al., 2003), such systems are easily adapted to ensure maximal use of detector time through staggered, parallel methods in which samples are extracted whilst previous extracts are being analysed (multiplexing). In this way, considerable savings in terms of time and solvent use can be achieved. In contrast with SPE, the extraction columns are re-usable for several hundred injections (Zeng et al., 2004; Chassaing et al., 2005). However, as with other automated methods, such equipment is associated with a high capital cost which hinders widespread uptake. A review by Xu et al. (2007) reveals many home-built systems for on-line SPE. The use in LLE of large volumes of toxic and environmentally polluting solvents has led to the development of many micro-extraction techniques. Minimal volumes and a small number of steps are typical. Analyte enrichment and recoveries are often high, but the methods are dicult to automate and generally involve a good deal of manipulation. A few namely restricted access materials (RAM), turbulent ow chromatography and micro-extraction by packed sorbent (MEPS) oer promise in terms of automation, solvent consumption, and ease of use (Novkov and Vlov, 2009). Many recent reviews of this emerging eld are available (Pedersen-Bjergaard and Rasmussen, 2008; Blomberg, 2009; Cruz-Vera et al., 2009; Kataoka, 2010; Sarafraz-Yazdi and Amiri, 2010; Vuckovic et al., 2010). More generally, analytes may be lost during hydrolysis of samples prior to analysis or extraction (Jourdil et al., 2003), and losses from the adsorption of analyte onto the walls of sample containers should always be checked for (Verplaetse and Tytgat, 2010).
Chromatographic Considerations
For LC-MS, the eluent composition corresponding to optimum analyte resolution does not always equate to that for optimal MS ionization of the analytes of interest. Non-volatile buers/eluent additives cannot be used, and strong acids such as triuoroacetic acid (TFA) may cause signicant signal suppression in positive ionization mode through ion-pairing of TFA anions with parent ions. The eect of eluent composition, additives, and adduct formation, on MS ionization has been reviewed extensively (Zhao et al., 2002; Mortier et al., 2004; Kostiainen and Kauppila, 2009; Table 5). The increasing demand for faster chromatography exacerbates the problem of co-eluting matrix components, since the most severe matrix eects occur early in the chromatographic run. Types of Column Ultra-high pressure liquid chromatography (UHPLC) using columns packed with sub-2 mm particles may shorten analysis time whilst retaining or improving chromatographic eciency (Nguyen et al., 2006), although ultra-high pressure LC pumps are necessary. A range of chemically modied stationary phases are available (Guillarme et al., 2010a). Such systems have been widely applied for high-throughput, targeted drug analyses. Eichhorst et al. (2009) report a semi-quantitative targeted screening analysis of 40 drugs/metabolites within 5.2 min, and a capacity for 200 urine samples per day. Berg et al. (2009) similarly describe the quantitative analysis of a series of opiates and cocaine/cocaine metabolites within 5.7 min. Matrix eects may be reduced when using UHPLC compared with HPLC, as interfering matrix components are more eciently separated from compounds of interest (Chambers et al., 2007). If UHPLC hardware is not available, supercially porous packing materials based on silica particles with non-porous cores may oer similar gains in eciency, but at column pressures within the range of standard HPLC pumps (Kirkland et al., 2007; Ali et al., 2010; Fekete et al., 2009). Rust et al. (2010) used a Phenomenex Kinetex (average 2.6 mm total particle
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Table 5. Summary of LC eluent considerations for LC-MS (and MS/MS) ionization Eluent composition Methanol, acetonitrile and aqueous eluents are most often used for both ESI and APCI. APCI is more amenable to non-polar solvents. Non-volatile buers (e.g. phosphates, borates) should be avoided. The most commonly used LC-MS eluent additives are formic acid, acetic acid, ammonium formate, and ammonium acetate. Eluent additives (e.g. ammonium, sodium, lithium, chloride, acetate) can produce adducts. This may be exploited, e.g. in the analysis of immunosuppressants such as sirolimus, which readily form adducts, or for compounds which themselves do not ionize readily. ESI is incompatible with high concentrations of eluent additives (>10 mmol/L). APCI can be used with much higher concentrations of additive. Water-rich eluent may not allow for the most ecient ionization. This is a problem for early-eluting analytes in reversed phase LC. HILIC may provide an alternative separation mechanism. Post-column addition of organic solvents may improve ionization eciency (Rentsch, 2003). Eluent pH may be manipulated to promote ionization in the eluent and hence improve ESI signal intensity. Eluent ow-rate APCI is more amenable to high ow-rates (>1 mL/min) than ESI. ESI can give increased MS signal intensity at lower ow-rates (0.1 mL/min or less, e.g. in capillary LC).
diameter) column for the separation of 21 benzodiazepines and three Z-Drugs in human hair. Monolithic columns may be useful alternatives for fast LC, by virtue of low back pressures, allowing high ow-rates (Berna et al., 2004; Guillarme et al., 2010a). Pihlainen et al. (2003) used a Chromolith C18 (Merck) monolith to identify and quantify 14 dierent compounds, including amphetamines, benzodiazepines, opiates and steroids, within 5 min. Temperature control for any LC separation is important for reproducible retention time data. This is especially true when considering screening analyses in which retention time is often used as a criterion for compound identication (Rivier, 2003; de Zeeuw, 2004). For thermally stable analytes, one may also consider high-temperature LC as a means of speeding up separations. Using such an approach, Nguyen et al. (2007) report the separation of nine doping agents in less than 1 min, using a sub2 mm column at 90C. An increased risk of column blockage associated with sub-2 mm packings is reduced by appropriate ltration of all mobile phases, and the use of in-line lters. Microbial growth in aqueous solutions can be reduced through regular renewal, or prevented by addition of a small amount of organic solvent. The latter also helps to reduce air-bubble formation when high-pressure mixing is used during gradient elution. The narrow peaks generated in such systems requires the MS cycle time/scan time to be suciently fast to ensure sucient data points are collected across chromatographic peaks. Stationary Phase Options The direct analysis of glucuronidated and sulfated urine conjugates of many drugs avoids the necessity for lengthy, and often poorly reproducible, enzymatic or chemical hydrolysis steps during sample preparation (Kaushik et al., 2006). These and other similarly polar analytes often pose problems when using traditional reversed-phase chromatography because the high aqueous content needed for adequate retention of these compounds may cause de-wetting (or phase-collapse). Moreover, for early eluting compounds, matrix eects caused by co-eluting matrix components may be more apparent. Polarembedded phases, or packings modied to allow 100%
aqueous eluents to be used are one way to overcome this issue. An alternative approach is that of hydrophilic interaction liquid chromatography (HILIC). HILIC phases (either bare silica or modied to contain a polar group, e.g. amide, cyano, diol or zwitterionic groups; McCalley, 2010) are now available in a number of particle sizes, including sub-2 mm and supercially porous. At low aqueous eluent composition, the formation of a water-rich layer close to the stationary phase facilitates separation through partitioning (hydrophilic interaction) of the analytes between this layer and the organic eluent component (Alpert, 1990; Hemstrom and Irgum, 2006). HILIC was used by Quintela et al. (2010) for the analysis of cocaine and metabolites (including cocaethylene) in hair, Al-Asmari et al. (2010) and Tarcomnicu et al. (2010) for the analysis of ethyl glucuronide, and Luiz Costa and Lanaro (2010) in the analysis of GHB in plasma and urine. There are a number of recent reviews and evaluations of HILIC materials (Chauve et al., 2010; Fountain et al., 2010; Jian et al., 2010; McCalley, 2010). Further, improved MS response was reported when using HILIC compared with reversed-phase LC (Grumbach et al., 2008). Peak tailing remains a potential problem for basic compounds on reversed-phase systems due to secondary interactions with residual silanol groups on the silica surface, and possible solutions are detailed in a recent review (McCalley, 2010). We have recently reported the application of a propylsulfonic acidmodied (strong cation-exchange, SCX) HPLC packing material (Couchman et al., 2010a) using 100% methanolic eluent for LC-MS/MS analysis of amphetamine, metamphetamine and amisulpride (Fig. 1). This simple, isocratic system is also suitable for analysis of a range of basic drugs. Non-silica based HPLC packings are rarely used in toxicological analyses, although Stephanson et al. (2002) showed application of a porous graphitic carbon column (Hypercarb, Thermo Scientic) for the analysis of ethyl glucuronide in urine. Kanno et al. (2009) used a thermoresponsive polymeric materialpoly(Nisopropylacrylamide)with a temperature gradient elution, for the quantitation of ve barbiturates in urine. MS is an achiral detection system. Hence, if chiral separations are necessary (for example amphetamine stereoisomers to determine pharmaceutical from street/clandestine amphetamine), LC
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Figure 1. (a) Amphetamine, metamphetamine and amisulpride in serum after liquidliquid extraction into butyl acetate : butanol (9 + 1, v/v); 20 mL of the extract was injected. HPLC: Waters Spherisorb S5SCX; 40 mmol/L ammonium acetate in methanol, pH* 6.0; ow-rate 0.5 mL/min. Detection: TSQ Quantum Access (Thermo Fisher Scientic). Results: amphetamine, 2 mg/L; metamphetamine, 197 mg/L; amisulpride, 242 mg/L. (b) Amphetamine and related phenethylamines. HPLC conditions: as above.
separation (using chiral LC columns, Kasprzyrk-Horden et al., 2010) or o-line stereoselective derivatization (Holler et al., 2005; Guillarme et al., 2010b) can be used.
Mass Spectrometry
When developing an LC-MS method, an important consideration is which type of atmospheric pressure ionization (API) to employ. This decision should be made based upon (i) the structure/ physiochemical properties of the analyte, (ii) the LC mobile phase composition (and ow-rate) at the expected time of analyte elution, (iii) knowledge of any drug metabolites which may or may not be chromatographically resolved from the analyte, and (iv) the sample preparation technique used. For the most part, the choice of ionization type will be between electrospray ioniza-
tion (ESI) and atmospheric pressure chemical ionization (APCI). Some modern instruments are supplied with dual-ionization sources, with the ability to rapidly switch between ESI and APCI during a chromatographic run (Waters ESCi; Gallagher et al., 2003) or even carry out both processes simultaneously (Agilent Multimode Source, Shimadzu DUIS 2010). Use of atmospheric pressure photoionization (APPI), as described by Robb et al. (2000), has not been widely reported for specic toxicological analyses, though is of potential use for very non-polar compounds in conjunction with very low ow-rate LC separations, which cannot be eciently ionized by APCI. For quantitative analyses, analyte ionization must be ecient and robust. Source conditions (temperatures, source gas owrates and voltages) which ensure complete desolvation reduce the risk of solvent cluster formation which can occur for some
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Table 6. Considerations for analyte optimization for LC-MS (and MS/MS) Optimize MS response for all analytes by infusion in eluent (including additives) rather than a dierent solvent. For gradient elutions, predict (or test if possible) mobile phase composition at the appropriate elution time, and use this mixture for analyte tuning. Always check for presence of adductscommonly occurring adducts include [M + Na]+ and [M + NH4]+. Check glassware and liquid handling apparatus as sources of possible adduct formation. Check for common in-source fragmentation (especially with APCI or H-ESI)e.g. [M + H - H2O]+. When optimizing analytes, whenever possible, separately infuse metabolites under the same conditions to check for in-source conversion back to parent compound, e.g. for N-oxides and S-oxides. In MS/MS avoid using commonly occurring fragments, such as dehydration or demethylation. Also try to avoid non-specic low m/z fragments. Use multiple SRM transitions whenever possiblethis can help dierentiate isobaric interferences, e.g. naloxone and 6-monoacetylmorphine. For multi-analyte methods, optimize the method for the analyte for which the highest sensitivity is needed, or which ionizes the least eciently.
analytes. Eorts to minimize, and/or correct for, suppression or enhancement of ionization as matrix components elute from the LC column must always be made. During tuning of the MS (Table 6), conditions should mimic, as closely as possible, those expected at the time of analyte elution, including concentrations of mobile phase additives (which may form adducts with the analytes), mobile phase composition and ow-rate. For multianalyte methods, tuning is often a compromise, and should be targeted towards the analyte of lowest concentration and/or ionization eciency. Electrospray Ionization Owing to the production of ions with multiple-charges, electrospray ionization (ESI) is useful for protein analysis and has been applied to the analysis of bioactive peptides (such as insulin and insulin-like growth factor) used in doping (Thomas et al., 2010a). In the eld of toxicology, for analysis of smaller molecules (around 1001000 Da), ESI is the most widely applied ionization technique. Indeed, many laboratories carrying out general unknown screening (GUS) or systematic toxicological analyses (STAs) utilize positive mode ESI in order to identify as many compounds as possible. It is suitable for analysis of highly polar compounds which are ionized in solution, such as glucuronide or sulfate conjugates in urine. However, reversed-phase LC generally results in early elution of highly polar compounds at correspondingly high aqueous mobile phase composition, which may result in reduced desolvation (hence ionization) eciency. To overcome this, some groups advocate postcolumn addition of organic solvent to improve desolvation of early-eluting compounds (Janda et al., 2002; Rentsch, 2003). In HILIC mode, polar compounds are eluted at high organic compositions and thus ionize more eciently (Nguyen and Schug, 2008). The use of high mobile phase electrolyte concentrations should be avoided when using ESI, due to the risk of suppression of ionization and contamination of the source. As a general rule, concentrations of volatile ionic modiers should remain below 10 mmol/L (Kostiainen and Kauppila, 2009). An important advantage of ESI is that signal intensity (as a function of the signal-to-noise ratio) is dependent not on the
absolute amount of analyte entering the source, but the concentration of the analyte (i.e. the concentration of analyte in the injected volume) and the eluent ow-rate (Polettini, 2006; Watson and Sparkman, 2007). Because of this, ESI is more applicable, and indeed gives increased sensitivity for some applications when LC separations are scaled down from 4.6 mm i.d. columns to 2.1 mm i.d. analytical columns, or from conventional HPLC to UHPLC. Nano- and capillary-LC applications are also reported for the analysis of toxicologically relevant compounds. Murphy et al. (2007) report the use of capillary LC for the analysis of nicotine and cotinine in plasma of smokers. The importance of complete desolvation means that ESI is generally limited to owrates of less than 1 mL/min. Flow-splitting is often used at higher ow-rates, since this has no eect on signal for a given concentration. Matrix eects are also reduced using this approach (Kloepfer et al., 2005). Heated-ESI (H-ESI) is now a further option from some MS vendors, which provides improved desolvation and, therefore, capacity for increased ow-rates through addition of a heated vapourizer. The thermal stability of analytes should be considered when using H-ESI. Atmospheric Pressure Chemical Ionization With atmospheric pressure chemical ionization (APCI), ionization occurs in the gaseous phase, making this type of ionization inherently more ecient than ESI for non-polar (hydrophobic) analytes, such as steroids, which do not readily form ions in solution (Maurer, 2007). As APCI rarely produces ions with multiple charges, the achievable mass range often equates to that of the operational range of the instrument. Since APCI requires use of a heated vapourizer, thermally labile compounds may decompose in the ionization source, nullifying the benet of ambient or sub-ambient separation achieved with LC. Indeed, certain compounds give rise to thermally labile N-oxide metabolites, which decompose in-source back to parent compound when using APCI (Peiris et al., 2004). In quantitative analyses where an N-oxide metabolite is present but not chromatographically resolved from the parent compound, this can lead to over-estimation of the concentration of the parent compound (Morgan et al., 2010; Fig. 2). Similarly, some glucuronide metabolites break down in
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Figure 2. Clozapine, norclozapine and clozapine N-oxide by TurboFlow-LC-MS/MS (0.50 mg/L each analyte in newborn calf serum, 10 mL directly injected onto TurboFlow column). Columns: TurboFlow, 50 0.5 mm Cyclone; analytical, ACE C18, 50 2.1 mm (3 mm average particle size).
source to form protonated aglycone pseudomolecular ions (Polettini, 2006). Further Considerations For most toxicological analyses using common LC mobile phases and columns, both ESI and APCI could be used interchangeably. With regards to quantitation, Beyer et al. (2007) demonstrated very similar accuracy and precision data when ESI was compared with APCI for the quantitation of a series of nine toxic alkaloids. Under the chromatographic conditions used, ESI achieved lower limits of detection than APCI. Despite optimization of LC and MS source conditions, some analytes will remain poorly ionized. In this situation, the use of chemical derivatization to generate species more amenable to ionization (for instance by addition of a proton-accepting moiety) is an option (Cech and Enke, 2001). Thieme et al. (2008) showed that improved sensitivity could be achieved for the analysis of buprenorphine and norbuprenorphine in plasma with formation of an N-methylpyridinium derivative. Derivatization often serves to increase the signal-to-noise ratio for analytes of lower molecular weight by increasing the observed m/z of the molecular ion species, to higher m/z values, where there is less interference.
Matrix Eects The eects of biological matrix components, such as proteins, lipids (notably phospholipids), sugars and salts, as well as other drugs/metabolites (including commonly encountered over-thecounter medications) and mobile phase components/sample preparation reagents on MS signal intensity are extensively reported and reviewed in the literature (Matuszewski et al., 1998; Fu et al., 1998; Annesley 2003; Souverain et al., 2004; Taylor, 2005; Matuszewski, 2006; Leverence et al., 2007; Ismaeil et al., 2008; Srinivas, 2009; Chambers, 2009; van Eeckhaut et al., 2009; Capiello et al., 2010; Gosetti et al., 2010; Marchi et al., 2010; Vogeser and Seger, 2010). Matrix eects may serve to increase (ion enhancement) or decrease (ion suppression) the MS signal, and can have profound eects on assay precision and accuracy in quantitative work. Concentrations of these non-detected interfering matrix compounds are often unknown, and highly variable between dierent samples/matrices. This degree of uncertainty is particularly true for the complex matrices of post-mortem forensic samples and has not been extensively studied in alternative matrices such as hair. Especially when considering fully quantitative analyses, it is therefore essential (and indeed has become a mandatory requirement when validating an bio-assay according
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L. Couchman and P. E. Morgan to the US Food and Drug Administration guidelines) to evaluate and attempt to minimize the incidence of matrix eects (FDA/ CDER, 2001; Peters et al., 2007). Consideration of sample collection and pre-treatment, as well as MS conditions, are essential. It is acknowledged that with regards MS conditions, ESI is more susceptible to matrix eects compared with APCI or APPI, and that ionization in the negative mode is more selective than the positive mode (LeBeau et al., 2000; Annesley, 2003; Dams et al., 2003b; Schuhmacher et al., 2003; Matuszewski, 2006; Maurer 2007; Flanagan et al., 2007; Smith et al., 2007). That said, matrix eects are known to occur and should always be evaluated for APCI methods as well (Sangster et al., 2004). Modern instruments tend to use a spray orthogonal to the entry orice of the mass spectrometer; hence only ionized species enter the MS, meaning higher ow-rates can be applied without signicant increases in noise (Niessen, 2003; de Homan and Stroobant, 2007). Two main methods exist for matrix eect evaluation, both of which are widely cited. The method proposed by Bonglio et al. (1999) uses post-column infusion of target analytes to qualitatively highlight regions of suppression/enhancement in a chromatogram, whilst that proposed by Matuszewski et al. (2003) suggests a method to quantify the degree of matrix eects for particular analytes using peak area/height ratios from analyses carried out with and without the presence of matrix components. Using both of these methods in combination allows for manipulation of chromatographic conditions and evaluation/ adaptation of sample pre-treatment procedures to minimize matrix eects. For exogenous compounds, analyte-free matrices obtained from dierent sources are recommended for thorough evaluation of matrix eects (as required in the FDA bioanalytical method validation guidelines; FDA/CDER, 2001). Matuszewski (2006) suggested that, for quantitative batch analyses, quality control samples prepared in dierent, independent matrices from the calibration standards should be included. It is well-documented that the use of stable isotope-labelled internal standards (ISTDs) serves to compensate for matrix eects (Matuszewski, 2006; OHalloran and Ilett, 2008; Tan et al., 2009; Marchi et al., 2010; Table 7). These isotopes should, in theory, exhibit identical behaviour to the analyte throughout the entire analytical procedure, including ion-suppression or enhancement eects. When the cost of these labelled compounds is prohibitive, for example in multi-analyte procedures, some groups have reported the use of analogous compounds as ISTDs; however in forensic cases, ingestion of the analogous compounds can never be unequivocally ruled out (Maurer, 2006). Moreover, recent reports of revised interest within the pharmaceutical industry in deuterium-substituted drugs may complicate the situation in the future. Drug analogues which are not licensed for treatment can usually be sourced from the drug manufacturers and other suppliers. Use of a representative ISTD for more than one analyte has also been reported (Liang et al., 2003; Remane et al., 2010). Whilst analogues may compensate for some matrix eects, labelled ISTDs have proved more useful and should be used wherever possible. However, Wang et al. (2007) described an example in which a deuterated ISTD (racaemic [2H5]-carvedilol) interacted dierently to the unlabelled analyte in dierent matrices. This study also suggested that ISTDs labelled with 13C, 15N or 17O may
Table 7. Internal standard considerations for LC-MS (and MS/MS) Use 13C, 15N or 18O-labelled ISTDs if possible. The ratio of the masses of these isotopes to 12C, 14N and 16O, respectively, is smaller than that of 2H to 1H, thus they will behave more similarly to the analyte. Also, these labels are often present at sites integral to molecular structure (e.g. 13C-enriched aromatic rings), and so are less likely than deuterated ISTDs to undergo exchange reactions. Despite this, deuterium-labelled ISTDs are the most commonly used. Ideally, use one ISTD for each analyte in multi-analyte methods. In MS/MS mode, try to use fragments which retain labelled atoms where possible. It it important to know the structures of labelled ISTDsthis may help identify the structure of fragments (e.g. for clozapine-D8, it can be deduced that fragmentation occurs as below).
Always evaluate matrix eects for ISTDs as well as analytes. Check whether the ISTD suppresses/enhances the analyte signal and vice versa. If optimizing MS response for stable isotope-labelled ISTDs, always check for the presence of unlabelled/partially labelled impurities. Whilst stable isotope labelled ISTDs may appear expensive, when calculated on a cost-per-test basis, they are often not prohibitively so. Stable isotope labelled ISTDs are not available for all compounds. Analogues of the analyte (sometimes available from pharmaceutical manufacturers) may be useful in these instances. Deuterated ISTDs may chromatographically separate from their unlabelled analogues, even under achiral LC conditions (Flanagan et al., 2007).
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LC-MS in analytical toxicology be better at compensating for matrix eects than deuterium labelled ISTDs (although they are often more costly). Likewise, Stovkis et al. (2005) and Lindegardh et al. (2008) emphasize that isotopically-labelled standards sometimes show dierent recoveries and chromatographic retention times relative to their un-labelled analogues. There is evidence that the ionization of some stable isotopically labelled ISTDs is itself suppressed or enhanced by the un-labelled analogues (Liang et al., 2003; Sojo et al., 2003; Maurer, 2005; Remane et al., 2010). It is good practice when optimizing MS conditions for a new labelled ISTD to check for unlabelled (or partially-labelled) impurities, and to select a fragment ion (or ions) retaining at least one isotopically-labelled atom where possible. A further consideration should be the matrix in which the ISTD is used. Addition to plasma of a relatively large proportion (20%, v/v) of acetonitrile containing ISTD resulted in a large number of matrix interferences when compared with lower proportions (Jemal et al., 2010). The inuence of the solvents used in sample preparation on matrix eects, and as a source of alkali metals as cations for adduct formation in some assays, has been reported (Annesley, 2007; Keller et al., 2008; Napoli, 2009). The volume (Liu et al., 2007) and composition of solvent mixtures (Zhang et al., 2008) used in sample preparation may signicantly aect the degree of matrix interference and should be evaluated during method development. For certain analytes, where sensitivity permits, dilution of matrix components as far as possible serves to reduce matrix eects (Schuhmacher et al., 2003; Kruve et al., 2009). Mei et al. (2003) highlighted the problems which can be caused by lithium-heparin containing sample collection tubes. Formation of [M + Li]+ adducts may aect both analyte recovery and chromatographic retention, and will lead to a reduced signal of the [M + H]+ ion. Similarly, Chin et al. (2004) reported reduced sensitivity to olanzapine when blood samples were collected in tubes containing potassium EDTA or sodium-heparin. The exact mechanism of adduct formation is not fully understood; however it has a signicant eect on quantitative LC-MS (Medvedovici et al., 2010). Mortier et al. (2004) concluded that a major concern is the reproducibility of adduct formation, particularly due to the variation in cation (Na+, K+, NH4+, etc.) concentrations in biological samples, and thus adduct ions should not ideally be used for quantitation. However, the use of stable isotope-labelled ISTDs was again advocated, as these should behave similarly to the analyte itself with regards to adduct formation. However, mobile phase additives (e.g. ammonium acetate) have been used to generate specic adduct ions for quantitation of certain compounds, notably immunosuppressants such as sirolimus and tacrolimus, which readily form adducts (Holt et al., 2000; Bogusz et al., 2007). Li et al. (2002) showed improved assay precision by monitoring and summing the signals of all adducts found compared with just monitoring a single adduct. However, as noted by Nozaki et al. (2010), this method assumes equal ionization eciency for all adduct forms. Sodiated adducts have been shown to produce dierent fragment ions than their relative protonated adducts (Nozaki et al., 2010; Medvedovici et al., 2010). Mass Analysis Mass analysers can be broadly classied into two groups: (i) scanning mass analysers (which only allow transmission of single m/z at a timethese include quadrupoles, ion-traps and magnetic sector instruments) and (ii) those which allow transmission of ions of diering m/z simultaneously (such as timeof-ight (TOF) instruments) (de Homan and Stroobant, 2007). The choice of mass analyser(s) will largely be decided by the analytical requirements and, of course, the capital cost of the instrument. For targeted, quantitative analyses, for instance in therapeutic drug monitoring (TDM) and targeted drugs of abuse analysis, LC-MS/MS is nowadays considered the method of choice. Triple quadrupole instruments are well-regarded as the gold-standard for such analyses, due largely to the ability to perform selective reaction monitoring (SRM) experiments. Rapid electronic control of the quadrupoles allows for many SRM experiments to be carried out very quickly, which is of advantage when considering accurate quantitation coupled with the sharp chromatographic peaks achievable with fast LC and UHPLC. A vast number of applications have been reported using triple quadrupole instruments in SRM mode. Reports of multi-targeted screening procedures using SRM mode LC-MS/MS are available (Gergov et al., 2003; Nordgren and Beck, 2004; Nordgren et al., 2005; Eichhorst et al., 2009). In developing quantitative, (multi-)targeted assays, it is important to consider the practical limitations of SRM-based analysis (Sauvage et al., 2008; Maurer, 2010). When just SRM scans are carried out, the absence of full-scan mass spectral data provides information relating only to the targeted compound(s). With this in mind, full-scan data may be useful in evaluating the eects of co-eluting, non-isobaric matrix components. Whilst SRM transitions are highly specic, numerous examples exist of isobaric interference from other compounds, particularly when only a single SRM transition per analyte is monitored. Allen (2006) reported interference during the analysis of tramadol by LC-MS/MS arising from ingestion of the antidepressant venlafaxine. Nordgren et al. (2005) reported a 35% false positive rate when monitoring 23 analytes using singletransition SRM mode. However, this improved signicantly upon addition of a second SRM transition. The use of multiple transitions for a single compound (and the ratio between the signal intensities for each) is, for this reason, commonplace (Kushnir et al., 2005; Concheiro et al., 2007; Sauvage et al., 2008). Interference may also arise from metabolites, or other compounds which fragment or thermally degrade in-source to form isobaric compounds (Peiris et al., 2004; Vogeser and Seger, 2010). We have reported such an example in the LC-MS/MS analysis of clozapine and norclozapine. Clozapine N-oxide, a minor plasma metabolite of clozapine, degrades under the APCI source conditions back to clozapine itself, making chromatographic resolution of these compounds essential (Morgan et al., 2010). SRM-mode, and the application of product ion ratios, is obviously limited for compounds which do not fragment reproducibly. A well-documented example is that of buprenorphine and norbuprenorphine. Whilst many laboratories overcome this issue by monitoring the non-fragmented pseudomolecular ion in quadrupole 3 (Q3), Ceccato et al. (2003) report that, by using collision pressure/energy at a level just below that which causes complete fragmentation of the precursor ion, isobaric interferences could be fragmented, thus increasing the observed signal-to-noise ratio. To minimize the risk of interference, multiple transitions (ideally avoiding non-specic transitions such as water loss(es) or low molecular weight fragments) should always be used where possible. For compounds which do not fragment well, or have
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L. Couchman and P. E. Morgan only one major fragment, extra steps should be taken to ensure selectivity during sample preparation and LC separation (Sauvage et al., 2008). Systematic Toxicological Analysis/General Unknown Screening In forensic and post-mortem toxicology, systematic toxicological analysis (STA)/general unknown screening (GUS) is the starting point from which further, targeted quantitative analyses follows (Flanagan et al., 2007), and aims to sensitively and reliably detect as wide a range of compounds as possible. It is the most crucial stage in forensic and post-mortem toxicological analysis, and to be as comprehensive as possible is a prerequisite. For many years GC-MS, despite the problems associated with larger, non-volatile and thermally labile compounds, was considered the best strategy for these analyses. The reproducibility of GC-MS ionization/ fragmentation allowed for the development of comprehensive mass spectra libraries for reliable structural identication. The lack of equivalent LC-MS based spectral libraries, which prevents universal adoption of this technique, is due to soft ionization achieved with API (compared with GC-MS ionization), and also the poor inter-instrument reproducibility of LC-MS fragmentation (Marquet, 2002; Kushnir et al., 2005; Jansen et al. 2005; Maurer and Peters, 2005;Yadav et al., 2008). Some groups however, report that reproducibility can be achieved with standardization of instrument settings (Bristow et al., 2004; Gergov et al., 2004; Hopley et al., 2008; Oberacher et al., 2009). Early STA methods using single-stage LC-MS (usually quadrupole) instruments, using in-source collision-induced dissociation (CID) to generate product ion mass spectra showed some promise, and users began to build considerable in-house spectral libraries of their own (Marquet et al., 2000; Hough et al., 2000; Lips et al., 2001; Saint-Marcoux et al., 2003; Venisse et al., 2003). However, LC-MS/MS (triple quadrupole or hybrid quadrupole-ion trap instruments) with information- or data-dependant acquisition is fast becoming considered a better way to perform STAs. Improved instrument scan rates and collision cell functions (for example collisional energy ramping and spectra averaging, enhanced product-ion scans, and reduction of space-charging eects) make for more product rich, reproducible mass spectra, which are then applicable toin-house reference libraries/databases (Marquet et al., 2003; Saint-Marcoux et al., 2003; Mueller et al., 2005; Dresen et al., 2006, 2009, 2010; Sauvage et al., 2006; Politi et al., 2007; Liu et al., 2009, 2010; Lynch et al., 2010; Maurer, 2010). An emerging approach to STA analysis is that of accurate mass (or exact mass, high-resolution) MS. By carrying out full-scan MS experiments at high mass accuracy (m/z up to 5 decimal places), it is possible to very precisely lter the full-scan data and extract analyte chromatograms with very low background noise. In this way, one can distinguish between compounds which have the same nominal, but dierent exact masses (e.g. benzoylecgonine and atropine, ecainide and diltiazem). High-resolution MS (HRMS) can be performed, as with quadrupoles and ion-traps, as either single-stage MS (Gergov et al., 2001; Pelander et al., 2003, 2008, 2010; Ojanper et al., 2005; Polettini et al., 2008; Lee et al., 2009) or as MS/MS (quadrupole-TOF, Q-TOF), allowing HRMS product ion scan data, with information-dependant acquisition (Pavlic et al., 2006; Decaestecker et al., 2004; Peters et al., 2010). TOF-HRMS is of particular interest in the application of empirical formula-based data libraries, with isotope pattern-matching software, and the potential to screen for unknown compounds (and identify their metabolites) by knowledge of elemental composition alone, without the absolute need for reference material (Ojanper et al., 2006). Exact mass identication of specic metabolites (Liotta et al., 2010) and systematic fragmentation (Q-TOF-MS) approaches (Tyrkko et al., 2010) have shown that even structural isomers can be distinguished using accurate mass. Further, retrospective interrogation of full-scan data can be useful to investigate the presence of new compounds/metabolites (such as new designer drugs) without re-analysis (Peters et al., 2010). Newer Orbitrap/Exactive technology (ThermoFisher Scientic), also capable of HRMS, is nding some toxicologically relevant applications (Thomas et al., 2010b; Weider et al., 2010; Johnson and Kozak, 2010) and may be a useful tool for the future. Where possible, for unequivocal compound identication, library matching should be carried out against databases produced in-house. New mass analysis technologies should be exploited whenever possible, in order to improve the production spectra data obtained, and thus create a more useful reference library. Mass spectra libraries and compound formulae databases from elsewhere may not be completely transferable between instruments (certainly with respect to product/ fragment ion ratios; Jansen et al., 2005) and library data will not give any indication of chromatographic retention time, which is a useful criterion for absolute identication (Rivier, 2003; Maralikova and Weinmann, 2004; de Zeeuw, 2004; Fox et al., 2009). Lynch et al. (2010) highlight the importance of manual interrogation of mass spectral data alongside automated library searching software to avoid false identications. Logistical Considerations Vogeser and Seger (2008) highlighted the considerable logistical requirements for installation of LC-MS instrumentation with regards to noise and space. A further important consideration relating to installation is that of ambient operating temperature. Many instruments are designed to operate optimally within a pre-determined temperature range. Exceeding these parameters may cause analytical problems, notably mass calibration drift. Placing instruments below air-conditioning vents, which may result in signicant temperature variation throughout the day, should therefore be avoided. Method Validation and Laboratory Accreditation The importance of analytical toxicology results necessitates reliable, accurate and often legally defensible results. A number of useful reports, ocial guidelines and reviews exist on the requirements and practicalities of bioanalytical method validation (Wood, 1999; Shah et al., 2000; FDA/CDER, 2001; Vander Heyden et al., 2001; Thompson et al., 2002; Taverniers et al., 2004; SOFT/AAFS, 2006; Viswanathan et al., 2007; EMEA/CHMP, 2009) and how these guidelines are best applied to clinical and forensic analytical toxicology (Peters and Maurer, 2002; Peters et al., 2007). The more recent of these guidelines/reviews highlight the need for matrix eect evaluation with LC-MS methods. For qualitative analyses, sensitivity (the ability to detect truly positive samples as positive), specicity (the ability to detect truly negative samples as negative) and limit of detection (LOD) should be ascertained at the very least, plus ideally investigations into analyte recovery, assay precision and robustness (Trullols et al., 2004; Peters et al., 2007). Forensic laboratory accreditation is based upon international standards (ISO/IEC 17025, 2005) which include requirements for method validation. The Society of
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LC-MS in analytical toxicology Forensic Toxicologists (SOFT) and the Toxicology Section of the American Academy of Forensic Sciences (AAFS) have issued useful guidelines for preparation of a laboratory for accreditation inspection (SOFT/AAFS, 2006).
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Conclusions
LC-MS and LC-MS/MS combine the versatility of HPLC with the sensitivity and selectivity of MS detection. This overcomes the limitations associated with GC-MS when analysing certain polar and non-volatile compounds, many of which are of signicance in toxicological investigations, but brings other problems. Advances in interface technology, and a better understanding of the complex physiochemical processes occurring during ionization, mean that LC-MS is becoming commonplace for routine, high-throughput, and specialist toxicological investigations. With regards to matrix eects, the importance of sample preparation and chromatographic separation cannot be over-emphasized. A number of approaches to minimizing matrix eects are available, and should form the basis of thorough method development.
Acknowledgements
The authors wish to thank Victoria Lay (Department of Chemistry, Loughborough University, UK) and Professor Robert Flanagan (Kings College Hospital, UK) for valuable assistance and suggestions, and also Dr Chang Kee Lim for the kind invitation to contribute to this Special Issue of Biomedical Chromatography.
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Special Issue: Review Article

(wileyonlinelibrary.com) DOI 10.1002/bmc.1566

LC-MS in analytical toxicology: some practical considerations

Copyright 2010 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 2011; 25: 100123

Biomed. Chromatogr. 2011; 25: 100123

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Copyright 2010 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 2011; 25: 100123

LC-MS in analytical toxicology

Biomed. Chromatogr. 2011; 25: 100123

Urine Urine Urine Plasma 1 mL/5 mL 1 mL/100 mL 100 mL/10 mL

Etizolam, brotizolam, lorazepam

Lee et al., 2003

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Allen et al., 2005

Mercerolle et al., 2008

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Sergi et al., 2010

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Methadone, EDDP, EMDP

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Biomed. Chromatogr. 2011; 25: 100123

LC-MS in analytical toxicology

Biomed. Chromatogr. 2011; 25: 100123

Laloup et al., 2005

Urine Post-mortem blood

ElSohly et al., 2006 Roman et al., 2008

Plasma Plasma 500 mL

Moody et al., 2002 Remane et al., 2010

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Diethyl ether. LOD 110 ng/mL

Deventer et al., 2006

Kala et al., 2008 Glover and Allen, 2010

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View this article online at wileyonlinelibrary.com

Copyright 2010 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 2011; 25: 100123

LC-MS in analytical toxicology

Biomed. Chromatogr. 2011; 25: 100123

Svensson et al., 2007 Parkin et al., 2008

Cannabinoids, opioids and stimulants (13 analytes)

MDMA and metabolites (3 analytes)

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Urine Urine Plasma

Langman et al., 2009 Robandt et al., 2008 Ashman et al., 2010

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Apollonio et al., 2006

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Various drugs (acidic, basic, polar, non-polar, aromatic; 11 analytes)

Biomed. Chromatogr. 2011; 25: 100123

Biomed. Chromatogr. 2011; 25: 100123

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L. Couchman and P. E. Morgan

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Biomed. Chromatogr. 2011; 25: 100123

LC-MS in analytical toxicology

Biomed. Chromatogr. 2011; 25: 100123