BIOMEDICAL CHROMATOGRAPHY

Biomed. Chromatogr. 21: 1077–1082 (2007)

Published online for
GC-MS method 21 June 2007 in Wiley
determination InterScience
of prilocaine HCl in human plasma ORIGINAL
ORIGINAL RESEARCH
RESEARCH 1077
(www.interscience.wiley.com) DOI: 10.1002/bmc.857

Development and validation of gas chromatography–mass

spectroscopy method for determination of prilocaine HCl
in human plasma using internal standard methodology

Yucel Kadioglu* and Alptug Atila

Department of Analytical Chemistry, Faculty of Pharmacy, Ataturk University, 25240 Erzurum, Turkey

Received 20 February 2007; revised 22 March 2007; accepted 23 March 2007

ABSTRACT: The accurate determination of prilocaine HCl levels in plasma is important in both clinical and pharmacological/
toxicological studies. Prilocaine HCl is quickly hydrolyzed to o-toluidine, causing methemoglobinemia. For this, the present work
describes the methodology and validation of a GC-MS assay for determination of prilocaine HCl with lidocaine HCl as internal
standard in plasma. The validation parameters of linearity, precision, accuracy, recovery, specificity, limit of detection and limit of
quantification were studied. The range of quantification for the GC-MS was 20–250 ng/mL in plasma. Within-day and between-
day precision, expressed as the relative standard deviation (RSD) were less than 6.0%, and accuracy (relative error) was better
than 9.0% (n = 6). The analytical recovery of prilocaine HCl and IS from plasma has averaged 94.79 and 96.8%, respectively.
LOQ and LOD values for plasma were found to be 20 and 10 ng/mL, respectively. The GC-MS method can be used for determi-
nation from plasma of prilocaine HCl in routine measurement as well as in pharmacokinetic studies for clinical use. Copyright ©
2007 John Wiley & Sons, Ltd.

KEYWORDS: prilocaine HCl; GC-MS; method validation

INTRODUCTION

Local anesthetic drugs are mainly used to reversibly

block nerve function in various local or regional treat-
ments. Local anesthetics play an important role clinically
in dentistry and minor surgery for temporary relief
of pain (Cawson and Curson, 1983; Rishiraj et al.,
2005). Prilocaine, 2-propilamino-N-o-tolil-propiyonamit
Figure 1. Chemical structure of prilocaine HCl.
hydrochloride (Fig. 1), and lidocaine HCl (Fig. 2),
2-diethylamino-N-(2,6-dimethyl fenil)-etanamid hydro-
chloride, are local anesthetic substances of the amide type
(Siluvera and Stewart, 1997). Prilocaine, unlike other
amide anesthetics, is a secondary amino derivative of
toluidine. It produces less vasodilation and toxicity than
lidocaine and is considered relatively free from allergic
reactions (Jastak et al., 1995). Prilocaine is extensively
metabolized by the liver. Prilocaine’s primary limiting
factor clinically is the production of methemoglobine- Figure 2. Chemical structure of lidocaine HCl.
mia, a side effect caused by its metabolite o-toluidine
(Warren et al., 1974; Ariens, 1989; Rudolf et al., 1995). specific analytical methods are needed for determina-
Low plasma concentrations of local anesthetic drugs tion of local anesthetics in human plasma. Several
are present after local injections, thus, sensitive and analytical methods for analysis of prilocaine in biologi-
cal samples have been reported. A chiral pressure
*Correspondence to: Y. Kadioglu, Department of Analytical Chem- liquid chromatography method for measure of the
istry, Faculty of Pharmacy, Ataturk University, 25240 Erzurum,
Turkey. separate isomers of prilocaine in plasma after adminis-
E-mail: yucel@atauni.edu.tr tration of the racemate (Tucker et al., 1990) and for the
Abbreviations used: LOD, limit of detection; QC, quality control. quantitation of R(−) and S(+) prilocaine in human
Contract/grant sponsor: University of Ataturk. serum (Siluvera and Stewart, 1996) has been reported.

Copyright © 2007 John Wiley & Sons, Ltd. Biomed. Chromatogr. 21: 1077–1082 (2007)
DOI: 10.1002/bmc
1078 ORIGINAL RESEARCH
An LC-MS-MS method for the simultaneous determi-
nation of the local anesthetics bupivacine, mepivacine,
prilocaine and ropivacine in human serum has been re-
ported (Koehler et al., 2005). Also, a stability-indicating
RP-HPLC method for determination of several procaine
hydrochloride and prilocaine hydrochloride combina-
tions has been reported (Storms and Stewart, 2002).
Watanabe et al. (1998) have analyzed five local anes-
thetics (lidocaine, dibucaine, mepivacaine, prilocaine
and bupivacaine) in human blood with headspace solid-
phase microextraction (HS-SPME) and gas chromato-
graphy–mass spectrometry–electron impact ionization
selected ion monitoring (GC-MS-EI-SIM) methods.
The determination of the binary mixture of prilocaine
and lidocaine in plasma, serum and pharmaceutical for-
mulations has been done with HPLC (Adams et al.,
1989; Klein et al., 1994; Taddio et al., 1997; Wieberg
and Jacobbssen, 2004; Wiberg et al., 2004) and a se-
quential injection chromatography method with a Franz
cell (Klimundova et al., 2006). Both drugs served as
internal standards for each other. Several studies with
GC methods have been reported for determination of
local anesthetics in biological samples (Keenaghan,
1967; Cameron, 1974; Asada, 1979; Prat and Brugerolle,
1986; Bjork et al., 1990). Prilocaine HCl has been
generally analyzed by combinations of several of local
anesthetic substances in biological materials. Therefore,
the purpose of this study was to develop and validate
a new GC-MS method for the determination of prilo-
caine HCl with a simple sample preparation without
derivatization in human plasma. The GC-MS method
has the advantage of specificity, simplicity, reproducibil-
ity and a wide range of linearity.
EXPERIMENTAL
Reagents and chemicals. Prilocaine HCl reference standard
and lidocaine HCl as internal standard (IS) was kindly sup-
plied by Novagenix Company and by the Turkish Doping
Control Center. The purity of these substances was checked
by standard methods (melting point, infrared and NMR spec-
tra) and no impurities were found. The high-purity grade
methanol and all other reagents were purchased from Merck
(Germany) and were used without further purification. All
gases were supplied by Havas (Ankara, Turkey). All solutions
were prepared daily.
Equipment. The GC/MS system Agilent 6890 series II gas
chromatograph, with an Agilent 7673 Autosampler, electronic
pressure control, an Agilent 7673 Autosampler and an Agilent
5972 series mass selective detector with electronic impact was
used. GC operating conditions were as follows: 5% methyl
phenyl silicone ultra 2 capillary column (12.5 m long, 0.2 mm
internal diameter and 0.33 µm film thickness, Hewlett Packard,
Waldbronn, Germany); the split mode (10:1) was used with
helium carrier gas and the flow rate of carrier gas was kept
constant during runs at 1.6 mL/min. The injector volume was
Copyright © 2007 John Wiley & Sons, Ltd.
Y. Kadioglu and A. Atila
3 µL. The injector and detector temperatures were 280°C. The
oven temperature programs were: initial temperature 90°C,
ramped at 10°C/min to a final temperature of 200°C and held
at this final temperature for 7 min. The MS detector para-
meters were: transfer line temperature 280°C; solvent delay
3 min; electron energy 70 eV; the MS was run in electron
impact mode with selected ion monitoring (SIM) for quantita-
tive analysis [m/z 81 for both prilocaine HCl and internal
standard (lidocaine HCl)].
Preparations of stock and standard working solutions.
Separate stock solution of prilocaine HCl and internal stand-
ard were prepared at a concentration of 100 µg/mL in metha-
nol and stored at −20°C when not in use. Prilocaine HCl
working solutions were daily prepared from stock solution up
to final concentrations of 20, 50, 100, 150, 200 and 250 ng/mL.
Internal standard working solution was prepared at the final
concentration of 500 ng/mL.
Preparation of plasma samples. The plasma samples were
stored at −20°C and allowed to thaw at room temperature
before processing. A suitable amount of working solution of
prilocaine HCl to a final concentrations from 20 to 250 ng/mL
and 0.2 mL of working solutions of IS was spiked in 0.5 mL of
blank plasma and then extracted with liquid–liquid extraction
method. The concentrations of the plasma standards (prilo-
caine HCl–IS mixtures) at respective points on the concentra-
tion graphs produced for plasma were 20, 50, 100, 150, 200
and 250 ng/mL prilocaine HCl together with 100 ng/mL IS at
each calibration level.
Quality control samples. Quality control (QC) samples
were prepared by adding aliquots of standard working solu-
tion of prilocaine HCl to a final concentrations of 40, 120 and
220 ng/mL, 0.2 mL of IS (100 ng/mL) and 0.5 mL of plasma
blank. The QC was prepared, spiked in plasma and extracted
following the procedure described below. These samples were
used in the analysis of plasma samples as quality controls for
the purpose of checking recovery of analyte in the daily
analyses of plasma samples.
Sample extraction. Prilocaine HCl was extracted from
plasma by developed liquid–liquid extraction. In 10 mL six
clean tubes were placed in 0.5 mL of plasma. Suitable
amounts of prilocaine HCl (20, 50, 100, 150, 200 and 250 ng/
mL) and 0.2 mL IS were then spiked into the plasma. The
solutions were alkalinized by shaking on a vortex mixer,
followed by addition of 1 mL of 1 M NaOH. A 5 mL aliquot
of diethylether solvent was added to the mixture, vortexed
for 5 min and then centrifuged at 4000g for 8 min. The or-
ganic layer was transferred into another tube and evaporated
to dryness at room temperature under nitrogen gas. The resi-
due was reconstituted with 100 µL methanol and vortexed for
30 s. A 3 µL aliquot was injected onto the GC-MS system.
Method validation. The validation of the method was carried
out by establishing specifity, linearity, recovery values, limits
of detection (LOD), limit of quantification, within- and
between-day precision and accuracy according to Interna-
tional Conference on Harmonization guidelines for validation
of analytical procedures (ICH, 1996).
Biomed. Chromatogr. 21: 1077–1082 (2007)
DOI: 10.1002/bmc
GC-MS method for determination of prilocaine HCl in human plasma
RESULTS
Specifity
The specificity of this method has been demonstrated
by checking the chromatograms obtained from the
plasma samples received from different hospitals. As
shown in Figs 3 and 4, good separation of prilocaine
HCl from internal standard was performed and no
further matrix interfering peaks at the retention times
of prilocaine HCl (tR = 9.10) and internal standard (tR =
9.50) in human plasma were observed. Different tem-
perature programs were investigated for exclusion of
matrix interference. At the end of this investigation,
the best temperature program was selected for a good
resolution and thus all the experiments used the oven
temperature program described above. When the ramp
rate was more or less than 10°C/min, good resolution of
the peaks (analyte peak and matrix interfering peaks)
was not obtained.
Figure 3. MS spectrum of prilocaine HCl.
Figure 4. GC-MS chromatograms of prilocaine HCl with lidocaine HCl as internal standard in
extracted plasma samples.
Copyright © 2007 John Wiley & Sons, Ltd.
ORIGINAL RESEARCH 1079
Linearity of calibration curves. Six standard samples
at different concentration levels ranging from 20 to
250 ng/mL with constant concentration of IS (100 ng/
mL) in spiked plasma samples were prepared and in-
jected onto the GC-MS system. The calibration (Fig. 5)
curve was established by plotting the ratio of the peak
areas of prilocaine HCl samples and the internal stand-
ard vs the concentration of prilocaine HCl obtained
after extraction of the spiked plasma sample. The linear
regression equation obtained using the least-squares
method (n = 6) and correlation coefficient are pre-
sented in Table 1. The mean regression equation is
y = 0.0049x − 0.0241 (y is the ratio of peak areas
of prilocaine HCl and IS, and x is the concentration
of prilocaine HCl). The calibration curve had excellent
linearity with correlation coefficient r = 0.9994.
Precision and accuracy. Assay precision was determined
by repeatability (within-day) and intermediate precision
(between-day). Within-day precision was evaluated by
Biomed. Chromatogr. 21: 1077–1082 (2007)
DOI: 10.1002/bmc
1080 ORIGINAL RESEARCH Y. Kadioglu and A. Atila

Table 1. Linearity of prilocaine HCl (n = 6)

Range LOD LOQ

Method (ng/mL) LR Sa Sb r (ng/mL) (ng/mL)
GC-MS 20–250 y = 0.0049x − 0.0241 0.0024 0.0105 0.9994 10 20

Based on six calibration curves. LR, linear regression equation; Sa, standard deviation of intercept of regression line; Sb, standard
deviation of slope of regression line; r, correlation coefficient.

be 20 ng/mL. Both accuracy and precision of this value

were well within the proposed criteria (RSD% < 20%).

Recovery. The analytical recovery of prilocaine HCl

Figure 5. Curve of calibration of prilocaine HCl in human
plasma.
assaying samples, six replicates set at the same concen-
tration (40, 120 and 220 ng/mL), during the same day.
The between-day precision was studied by comparing
the same assays on different days (10 days). The accu-
racy of this analytic method was evaluated by checking
at three different concentrations (QC) of prilocaine
HCl and 100 ng/mL of IS. The relative standard
deviation (RSD) values for within-day and between-
day precision were <4.0 and <6%, respectively (n = 6).
Precision studies of GC-MS method showed acceptable
RSD values, and relative errors for both within-day and
between-day accuracy were <3.5 and <9.0%, respec-
tively. These results are given in Table 2.
Limits of detection and quantification. The limit of
detection (LOD), defined as signal/noise = 3, was found
by analyzing a series of standard solutions containing
decreasing amounts of prilocaine HCl for plasma sam-
ples (RSD > 20% for LOD) to be 10 ng/mL. The limit
of quantification (LOQ), defined as signal/noise = 10 in
the lowest concentration of the prilocaine HCl for
plasma samples (RSD > 10% for LOQ), was found to
from human plasma was assessed by comparing the
concentration of the extracted samples with the concen-
tration of standard prilocaine HCl samples. The con-
centrations of prilocaine HCl in spiked plasma were 20,
163, 50, 100, 150, 200 and 250 ng/mL in the calibration
graph. Prilocaine HCl was extracted from blood with
5 mL diethylether solvent. This solvent gave an excel-
lent recovery. Analytical recoveries of prilocaine HCl
from plasma were 92.55–98.79%, as shown Table 3.
The recovery of IS from plasma in the same extracted
method was found to be 96.8%. Figure 4 shows a
chromatogram of solutions obtained from plasma
at concentrations of 20, 50, 100, 150, 200 and 250 ng/mL
of prilocaine HCl and 100 ng/mL of lidocaine HCl as
internal standard. This chromatogram was free from
interferences.
Interference study. In order to assess the possible
analytical applications of the proposed method, the
effect of common drugs used for patients was studied
by analyzing spiked plasma samples. The absence of
interfering compounds was verified by comparison
of chromatograms of plasma samples with those of
calibration plasmas. To test of the effect of the drugs
carbamazepine, gemcitabine, gentamisin, procaine HCl
and sevoflurane, and vitamin E and a multivitamin
tablet (Megadyn® film tablets containing 10,000 IU
vitamin A, 20 mg vitamin B1, 5 mg vitamin B2, 11.6 mg
calcium D-pantothenate, 10 mg vitamin B6, 0.005 mg
vitamin B12, 150 mg vitamin C, 500 IU vitamin D3,
10 mg vitamin E, 0.25 mg Biotin, 50 mg nicotinamide,

Table 2. Precision and accuracy of prilocaine HCl from six independent sets of spikes plasma samples

Within-day Between-day
Added Found ± SD Precision, Accuracy Found ± SD Precision, Accuracy
(ng/mL) (n = 6) %RSD (%) (n = 6) %RSD (%)
Plasma poolsa
40 38.7 ± 1.5 3.87 −3.25 36.4 ± 2.1 5.77 −9.00
120 115.9 ± 2.2 1.90 −3.42 113.5 ± 2.7 2.38 −5.42
220 214.7 ± 2.5 1.16 −2.41 210.5 ± 3.8 1.81 −4.32

SD, standard deviation of six replicate determinations; RSD, relative standard deviation.
a
Plasma volume, 0.5 mL.
Accuracy = %relative error = (found − added/added)100.

Copyright © 2007 John Wiley & Sons, Ltd. Biomed. Chromatogr. 21: 1077–1082 (2007)
DOI: 10.1002/bmc
GC-MS method for determination of prilocaine HCl in human plasma
Table 3. Accuracy and recovery data of prilocaine HCl which were obtained from
assay for added concentrations (n = 6)
Added Measurement
Method (ng/mL) (ng/mL)
GC-MS 20 18.51
50 46.71
100 94.90
150 141.38
200 189.68
250 246.97
25.8 mg phosphorus, 10 mg Fe, 50 mg Ca, 36.2 mg Mg,
0.5 mg Mn, 1 mg Cu, 0.5 mg Zn and 0.1 mg Mo), we
spiked solutions of these drugs (100 ng/mL) together
with 100 ng/mL prilocaine HCl solution in plasma,
mixed and then extracted them. The peak areas of the
extracted sample solutions were measured. The toler-
ance was defined as the concentration of the added
substance causing a relative error less than 3%. In the
case of solutions of these drugs we did not notice any
interference.
DISCUSSION
Local anesthetics are widely used for various local or
regional treatments. These drugs cause occasional
medical accidents. An accurate, simple and rapid
method for analysis of local anesthetics is required
in forensic practices (Watanabe et al., 1998). Total
and free fraction plasma and serum levels of local
anesthetics must measure for each drug alone and a
correlation between the drug concentrations. The
cardiovascular system toxicity of these drugs has been
reported (Koehler et al., 2005). These reports have
led to a renewed interest for determination of local
anesthetics. Although the local anesthetic prilocaine is
less cardio- and neurotoxic than lidocaine, it bears the
disadvantage of the formation of methemoglobin by the
metabolite o-toluidine (Warren et al., 1974; Tucker
et al., 1990; Rudolf et al., 1995). Thus, sensitive and spe-
cific analytical methods are needed for determination
of prilocaine in plasma after local injections. GC-MS is
powerful technique for highly specific and quantitative
measurements of low levels of analytes in biological
matrices. There have also been several other reports on
the use of liquid chromatography–tandem spectrometry
technique and HPLC and GC methods to measure
local anesthetics in human plasma. An extensive survey
of the literature showed that no GC-MS method for
the analysis of prilocaine HCl alone in human plasma
has been reported. In the present study, we report a
highly selective gas chromatography–mass spectrometry
(GC-MS) method for determination of the prilocaine
HCl of substances used as local anesthetics without
Copyright © 2007 John Wiley & Sons, Ltd.
ORIGINAL RESEARCH 1081
Accuracy Recovery
(%) (%)
−7.45 92.55
−6.58 93.42
−6.50 94.90
−5.75 94.25
−5.16 94.84
−3.21 98.79
derivatization in human plasma. A liquid–liquid extrac-
tion method developed from drug-spiked plasma
samples of prilocaine HCl was used. Under the used
conditions one single peak was present in the GC
chromatogram identified on the basis of two character-
istic ions at m/z 86 (base ion) and 107, shown in the
mass spectrum. This procedure was simple. Only a
small sample volume of 0.5 mL plasma was necessary
to achieve a limit of quantification of 20 ng/mL. The
proposed method can be conveniently used for the rou-
tine measurements of prilocaine HCl in plasma samples
and quality control laboratories, where economy and
time are essential.
CONCLUSION
This assay method for determination in human plasma
of prilocaine HCl from local anesthetic substances was
completely validated using sensitivity, stability, specificity,
linearity, accuracy and precision parameters. The GC-
MS method has high recovery and excellent reproduc-
ibility. For this reason, it can be used for determination
from plasma of prilocaine HCl in routine measurement
as well as in pharmacokinetic studies for clinical use.
Acknowledgments
The authors are grateful to the Directorship of the Univer-
sity of Ataturk for the financial support of this work.
REFERENCES
Adams HA, Biscoping J, Ludof K, Borgmann A, Beckman-MB and
Hempelmann G. The quantitative analysis of amide local
anesthetics using high pressure liquid chromatography and ultra-
violet detection (HPLC/UV). Regional Anaesthesia 1989; 12: 53 –57.
Ariens EJ. Chiral Separation in HPLC, Applications to Pharmaceuti-
cal Compounds, Krstulovic AM (ed.). Ellis Horwood: Chichester,
1989; 31.
Asada A. Gas chromatographic determination of local anesthetics in
blood. Osaka City Medical Journal 1979; 25: 91–122.
Bjork M, Petterson KJ and Osterlof G. Capillary gas chromato-
graphic method for simultaneous determination of local anesthetics
in plasma samples. Journal of Chromatography 1990; 533: 229–
234.
Biomed. Chromatogr. 21: 1077–1082 (2007)
DOI: 10.1002/bmc
1082 ORIGINAL RESEARCH
Cameron JD. The gas chromatographic determination of plasma
concentrations of some local anesthetics using a nitrogen detector.
Clinical Chemica Acta 1974; 56: 307–309.
Cawson RA and Curson DR. The hazards of dental local anesthetics
in children. British Dental Journal 1983; 154: 253–256.
Jastak JY, Yagela JA and Donaldson D. Local anesthesia in the oral
cavity. In: Pharmacology of Local Anesthesia and Clinical Prepara-
tions and Drug Selection. W.B. Saunders: Philadelphia, PA, 1995;
23 –26.
Keenaghan JB. The determination of lidocaine and prilocaine in
whole blood by gas chromatography. Journal of Pharmacy Science
1967; 56: 1645 –1646.
Klein J, Fernandes D, Gazarian M, Kent G and Koren G. Simultane-
ous determination of lidocaine, prilocaine and the prilocaine
metabolite o-toluidine in plasma by high performance liquid
chromatography. Journal of Chromatography B 1994; 655: 83 –88.
Klimundova J, Satinsky D, Sklenarova H and Solich P. Automa-
tion of simultaneous release tests of two substances by sequential
injection chromatography with Franz cell. Talanta 2006; 69: 730–
735.
Koehler A, Oertel R and Kirch W. Simultaneous determination
of bupivacaine, mepivacain, prilocaine and ropivacain in human
serum by liquid chromatography–tandem mass spectrometry. Jour-
nal of Chromatography A 2005; 1088: 126 –130.
Prat M and Bruguerolle B. Rapid simultaneous gas–liquid chromato-
graphic determination of five local anesthetic drugs in serum. Clini-
cal Chemistry 1986; 32: 2098 –2100.
Rishiraj B, Epstein JB, Fine D, Nabi S and Wade NK. Permanent
vision loss in one eye following administration of local anesthesia
for a dental extraction. International Journal of Oral & Maxillofa-
cial Surgery 2005; 34: 220–223.
Rudolf B, Durstewitz-Knierim D, Ridderskamp I, Scharenberg C
and Brandt L. Increase of prilocaine-induced methemoglobinemia
following anesthesia. Anaesthetist 1995; 44: 445–449.
Siluveru M and Stewart JT. Stereoselective determination of R-(−)-
and S-(+)-prilocaine in human serum using a brush-type chiral
Copyright © 2007 John Wiley & Sons, Ltd.
Y. Kadioglu and A. Atila
stationary phase, solid-phase extraction and UV detection. Journal
of Pharmaceutical and Biomedical Analysis 1996; 15: 389–392.
Siluvera M and Stewart JT. Stereoselective determination of R-(−)-
and S-(+)-prilocaine in human serum by capillary electrophoresis
using a derivatized cyclodextrin and ultraviolet detection. Journal
of Chromatography B 1997; 693: 205–210.
Storms LM and Stewart JT. Stability-indicating HPLC assay for deter-
mination of prilocaine and procaine drug combinations. Journal of
Pharmaceutical and Biomedical Analysis 2002; 30: 49–58.
Taddio A, Stevens B, Craig K, Rastogi P, Ben-David S and Shennan
A. Efficacy and safety of lidocaine–prilocaine cream for pain dur-
ing circumcision. New England Journal of Medicine 1997; 336:
1197–201.
Tucker GT, Mather LE, Lennard MS and Gregory A. Plasma con-
centrations of the stereoisomers of prilocaine after administration
of racemate: implications for toxicity? British Journal of Anaesthe-
sia 1990; 65: 333–336.
Warren RE, Van de Mark TB and Weinberg S. Methemoglobinemia
induced by high doses of prilocaine. Oral Surgery Oral Medicine
Oral Pathology 1974; 37: 866– 871.
Watanabe T, Namera A, Yashiki M, Iwasaki Y and Kojima T.
Simple analysis of local anesthetics in human blood using
headspace solid-phase microextraction and gas chromatography–
mass spectrometry–electron impact ionization selected ion monitor-
ing. Journal of Chromatography B 1998; 709: 225–232.
Wieberg K and Jacobbsson SP. Paralel factor analysis of HPLC-
DAD data for binary mixtures of lidocaine and prilocaine with
different levels of chromatographic separation. Analytica Chimica
Acta 2004; 514: 203–209.
Wieberg K, Andersson M, Hagman A and Jacobsson SP. Peak
purity determination with principal component analysis of high-
performance liquid chromatography–diode array detection data.
Journal of Chromatography A 2004; 1029: 13 –20.
ICH. Validation of analytical procedures. In Proceedings of the
International Conference on Harmonization. Commission of the
European Communities: Brussels, 1996.
Biomed. Chromatogr. 21: 1077–1082 (2007)
DOI: 10.1002/bmc