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Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/ijcas

Original Article

A rational approach for HPLC calibration using

mass spectrometer as detector
Paresh Varshney a,*, Atul Kaushik b, Alok Sharma c, Sajal Srivastava d,
Parmesh Kumar Dwivedi d
a

Department of Pharmacology, Sai Nath University, Ranchi, Jharkhand 835217, India

Formulation and Development, Watson Pharma Pvt Ltd., Mumbai 400099, India
c
Analytical Division, Dabur Research & Development Centre, Dabur India Ltd., Sahibabad 201010, India
d
Amity Institute of Pharmacy, Amity University Uttar Pradesh, Lucknow Campus Gomti Nagar Extension,
Lucknow 226010, Uttar Pradesh, India
b

article info

abstract

Article history:

Background/Objective: A direct, accurate, precise, reproducible and cost-time efficient

Received 25 February 2013

approach for calibration of HPLC using mass spectrometer as detector has been described

Accepted 2 April 2013

which simulates the routine analysis in bioanalytical laboratories.

Available online 9 April 2013

Methods: Injector position check and injection volume repeatability test for the calibration
of autosampler; and pump flow rate accuracy test for the calibration of pumps were

Keywords:

illustrated using direct weighing method. Carryover test, pump repeatability test, linearity

HPLC calibration

and precision were carried out for holistic (overall system) calibration of the instrument

LCeMS/MS

using a sensitive high throughput LCeMS/MS pre-validated method.

Injection volume repeatability test

Results/Conclusion: The results of calibration experiments for individual modules suggested

Injector position check

that pumps are yielding accurate flow rate, autosampler is aspirating the correct injection

System suitability

volume irrespective to position or volume, and column oven and autosampler chambers
are maintaining steady temperatures. Holistic calibration verified the accuracy, precision,
repeatability and reproducibility of the instrument with insignificant carryover.
Copyright 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights
reserved.

1.

Introduction

High-performance liquid chromatography coupled to mass

spectrometry (LCeMS/MS) has been the mainstay of bioanalytical industry from last two decades.1e4 It amalgamate
the chromatographic resolution power of HPLC and the
sensitivity and specificity of mass spectrometer to develop
rapid and high throughput methods for the bioanalysis of

small molecule drugs, metabolites and endogenous compounds in biological matrices.4e7

The bioanalytical laboratories throughout the world rely on
conventional procedures for HPLC calibration which use
Photodiode Array (PDA) or variable UV/Visible spectrophotometers as detectors.8e14 However, due to continuous evolution of bioanalytical techniques and with the rising
compliance expectations, an increased trend has been

* Corresponding author. Bioanalytical Department, DX Clinical, 260, Zone Industrial, El Jadida 24000, Morocco. Tel.: 212 653686403;
fax: 212 523373127.
E-mail address: varshneyparesh@gmail.com (P. Varshney).
0976-1209/$ e see front matter Copyright 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ijcas.2013.04.001

i n t e r n a t i o n a l j o u r n a l o f c h e m i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 1 0 2 e1 0 7

observed for the number of FDA 483 observations for

HPLC calibration while using hyphenated LCeMS/MS instruments.15,16 The limitations to the conventional approaches for HPLC calibration are summarized below.
The assessment of injector position check (IPC) and injection volume repeatability test (IVRT) is done by statistical
comparison between peak area response and the concentration of the injected samples from different vial positions
(for IPC) or with different volume injections (for IVRT).
Usually the precision (for IPC and IVRT) and coefficient of
correlation (for IVRT) are calculated for results interpretation. However, the procedures count on the indirect measurement of peak area response which is the function of
detector and not the autosampler. This indirect approach
for autosampler calibration cannot express the results
accurately especially when the autosampler is compromised due to unnoticed hardware alterations or aging
components.
Second limitation is the irrational coupling of HPLC to low
sensitivity UV detector for holistic (overall) calibration. As
bioanalytical methods involve analysis of drugs at very low
concentrations, it is essential to ensure that even the slight
variations in the performance of HPLC are noticed by the detector during calibration which may not be possible by UV
detector because of its low sensitivity.
Moreover, in the straightforward procedures like
measuring the effluent volume for pump flow rate accuracy
(PFRA) or temperatures for column oven or autosampler no inprocess evidence documents are generated.
The proposed research work is aimed to demonstrate
methodologies for the calibration of HPLC using highly sensitive mass spectrometer as detector to overcome the limitations of the conventional procedures. All the modules of HPLC
viz. pumps, column oven and autosampler were individually
calibrated without using any detector and then holistic calibration was performed coupling HPLC in-tandem with mass
spectrometer.

2.1.

Materials and methods

A selective, sensitive and validated LCeMS/MS bioanalytical

method for the estimation of Pioglitazone (PIO) was used as a
model to carry out HPLC calibration. The method was validated over the concentration range of 24.00e4003.30 ng/ml for
PIO using Metaxalone (MET) as an Internal Standard (IS).

Chemicals and reagents

Pioglitazone hydrochloride (purity: 98.90%) and metaxalone

(purity: 99.89%) were sourced from Varda biotech (P) limited
(Mumbai, India). HPLC-grade acetonitrile was sourced from
Rankem (India) and methanol was sourced from S.D. fine chem
limited (Mumbai, India). HPLC grade water was obtained from
a Milli-Q gradient A10 purifier. Ammonium formate (analytical
grade) was sourced from Panreac (Barcelona, Spain).

2.2.

Instrumentation

Holistic calibration was carried out on Perkin Elmer HPLC (series 200 LC pumps, series 200 column oven, series 200 autosampler) connected in-tandem with calibrated API-3200 triple
quadrupole mass spectrometer (from MDS Sciex, Applied Biosystems, Canada). Gas generator (from Peak Scientific, Scotland), Model NM20ZA was used to supply nitrogen gas and zero
grade air. The instrumentation was monitored using Analyst
software version 1.5 for data acquisition and processing.

2.3.

LCeMS/MS conditions during holistic calibration

An isocratic mobile phase {ammonium formate buffer 5 mM

(pH 6.2): acetonitrile (10:90, v/v)} was run on Waters XTerra MS
C18 5 mm (4.6 mm  150 mm) column at a constant flow rate of
0.5 ml/min. The temperatures of the column oven and autosampler were maintained at 40  C  1.0  C and 10  C  1.0  C,
respectively.
Mobile phase was introduced into mass spectrometer
through an ESI source operating in the positive mode at an ion
spray voltage of 5000 V. The transitions of PIO and MET were
monitored at 357.10 > 134.10 and 222.20 > 161.20, respectively.
The source and compound parameters optimized for the
detection are summarized in Table 1.

2.4.

2.

103

Calibration parameters

The calibration of HPLC was performed in the following

order: Pumps {PFRA test} / Column Oven {COTP
check} / Autosampler {ASTP check, IPC (for precision), IVRT
(for precision and linearity)} / holistic calibration {system
suitability, precision, linearity}. Before calibration, each
module was shutdown and powered-up to evoke automated
internal initialization procedure and built-in diagnostics for
detecting problem situations.

Table 1 e Optimized parameters for mass spectrometry.

Source and gas parameters
Curtain gas (psi)
Ion spray voltage (volts)
Heater gas (psi)
Source temperature ( C)
Nebulizer gas (psi)
Collision associated dissociation gas (psi)

Compound parameters
25
5000
50
550
50
6

Declustering potential (volts)

Entrance potential (volts)
Collision energy (volts)
Collision cell exit potential (volts)
Dwell time (msec)

PIO
80
5
25
3
200

MET
32
10
14
3
200

104

2.5.

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PFRA test

PFRA test was performed separately on each of the two pumps

of HPLC at 0.2, 0.5, 1.0, 2.0 ml/min of flow rate. The Milli-Q
water effluent was collected in a dry volumetric flask for
5 min. The volumetric flask was weighed before and after the
effluent collection, in grams. % Deviation of actual flow rate
should be within 2.0% of the set flow rate17e20 and was
calculated as per the following formula:
% Deviation Mean of actual flow rate=Set flow rate
 1  100

2.6.

COTP and ASTP check

COTP check was carried out at temperature settings of 30  C

and 50  C while ASTP check was carried out at temperature
settings of 5  C and 10  C. Two readings were recorded, each at
a gap of at least 15 min, for all temperature settings. The difference between the mean of actual temperature and set
temperature should typically be within 2  C.17,18

2.7.

Injector position check

Milli-Q water was transferred to five different vials, weighed

in grams, and placed in the autosampler at 1, 10, 55, 91 and 100
vial positions to cover the entire sample area. One injection of
10 ml was performed from each vial and the vials were weighed
again. The whole procedure was performed three times.
Actual injection volume was calculated as follows:
fWeight of vial before injection
 Weight of vial after injectiong  1000
Mean injection volume, standard deviation and coefficient
of variation (%RSD) were calculated at every vial position.
Global %RSD was also calculated for the injection volume. %
RSD should be <2%.17e19,21

2.8.

Injection volume repeatability test

Milli-Q water was transferred to five different vials, weighed

in grams, and placed in the autosampler. Triplicate injections
of 5 ml from first, 10 ml from second, 15 ml from third, 20 ml from
fourth and 25 ml from the fifth vial were performed and the
vials were weighed again. Actual injection volume (ml) at each
injection level was calculated as follows:

and the set injection volumes (x-axis). Linear coefficient of

correlation should be >0.99 while %RSD should be <2% for
injection volume accuracy between different injection
levels.17,18

2.9.

Holistic calibration

System suitability was performed to evaluate carryover,

pump repeatability and overall system verification using
both pumps and connecting HPLC in-tandem to mass spectrometer. An aqueous mixture of PIO (60.00 ng/ml) and MET
(170.00 ng/ml) was prepared in the mobile phase. 8 injections
were carried out on LCeMS/MS in the following order: one
injection of mobile phase blank (BLK1) / 6 injections of
aqueous mixture (AQMIX) / second injection of mobile phase
blank (BLK2).
Carryover for PIO and MET was calculated as follows:
fResponse of PIO or MET in BLK2
 Response of PIO or MET in BLK1g=
fMean peak area response of PIOor METin AQMIXg  100
Carryover should be 0.1% for both PIO and MET. Moreover,
the response of PIO (or MET) in BLK1 and BLK2 should not
exceed 5% of the mean peak area response of PIO (or MET) in
AQMIX.17e21
Pump repeatability performance was checked by %RSD of
the RT of PIO and MET which should be within 5%. %RSD for
peak area response ratio of PIO to MET should be 4% as a
mark of overall system verification.17,18
To evaluate linearity and precision for overall system
verification, the standard stock solution for PIO was prepared
in methanol with 1 mg/ml concentration and stored at
refrigerated temperature between 2 and 8  C. The stock was
further sequentially diluted using methanol:water solution
(50:50, v/v) and spiked into mobile phase to obtain a calibration curve ranging from 24.00 to 4003.30 ng/ml. Calibration
curve was consisted of eight non-zero standards, each mixed
with a constant concentration of MET (IS), and injected in
replicates of three. The concentrations of calibration standards were back calculated from a linear fit regression curve
between peak area ratio of PIO to MET (y-axis) and concentration (x-axis) using 1/1/x2 as the weighted scheme. The
coefficient of correlation was determined from all three calibration curves. The precision was calculated using three
calibration curves and should be <5% for each calibration
standard. The coefficient of correlation should be 0.99 for
each calibration curve.17e19

fWeight of vial before injection

 Weight of vial after injectiong  1000=3
Injection volume accuracy at each injection level was also
calculated as follows:
fActual injection volume ml=Set injection volume
i:e: 5; 10; 15; 20 or 25 mlg  100
IVRT was evaluated by determining %RSD for injection
volume accuracy between different injection levels and coefficient of correlation by plotting a regression curve between
Actual Injection Volume at different injection levels (y-axis)

3.

Results and discussion

The conventional approach of calibrating HPLCs coupling to

UV detector is a time consuming procedure and typically take
2e3 days of an analyst to track down the number of apparatuses, prepare the standards and dilutions number of times,
perform the experiments and document the results in the
laboratory notebook. This calibration time, when multiplied
by more than 25 HPLC systems twice a year, translated into
thousands of nonproductive hours. In our bioanalytical

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Table 2 e Results for PFRA, IPC and IVRT.

PFRA Results
Set flow rate (mL/min)

Mean flow rate (mL/min)

% Deviation

Mean flow rate (mL/min)

Pump 1
0.2
0.5
1.0
2.0

Pump 2

0.200954
0.50324
1.005985
1.983424

0.48
0.65
0.60
0.83

IPC Results
Vial Position
1
10
55
91
100
Mean global (mL)
%RSD global

% Deviation

0.201691
0.504815
0.991259
1.988246

0.85
0.96
0.87
0.59

IVRT Results
Mean injection
volume (mL)
9.88
9.98
9.95
10.00
9.96

 0.04
 0.11
 0.03
 0.11
 0.09
9.95 0.08
0.80

%RSD

Set injection
volume (mL)

0.44
1.06
0.25
1.05
0.86

5
10
15
20
25
Mean injection volume accuracy
Coefficient of correlation (R2)

laboratory, the holistic calibration of HPLC was completed in a

day using in-tandem mass spectrometer as detector with a set
of meaningful acceptance criteria that conform to the industry norms.
For PFRA test, % deviation found to be less than 1% for both
the pumps at the set flow rates of 0.2e2.0 ml/min using direct
weighing method (Table 2). The method proposed less margins of error as high sensitivity analytical balance was used to
mark the volume measurement. The process was time efficient. In-process data was also generated on the analytical
balance.
For COTP check the deviation in temperatures ranged from
0.07 to 0.13  C while for ASTP check the deviation was
0.00e0.03  C from the set temperatures. Datalogger was used

Actual injection
volume (mL)

Injection volume
accuracy

5.01
10.02
15.05
20.00
24.97

100.20
100.17
100.36
99.98
99.89
100.12  0.18
1

to record the temperature readings as a means of generating

in-process document.
To calibrate autosampler, IPC and IVRT were performed
using direct weighing method. %RSD ranged from 0.25 to 1.06
at individual positions while %RSD for injection volume at all
the positions (global statistics) found to be 0.80 during
IPC (Table 2). The calibration plot for IVRT was generated between actual injection volume and set injection volume at
different injection levels and yielded a linear equation
(y 0.9981x0.038) with a unit coefficient of correlation. The
precision for IVRT was evaluated in terms of %RSD for injection volume accuracy between different injection levels and
found to be 0.18 (Table 2). As IPC and IVRT were executed
without any detector, completed with ease in few hours, and

Fig. 1 e Representative LCeMS/MS chromatograms of BLK1, AQMIX and BLK2 of PIO and MET for carryover test.

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Table 3 e Results for holistic calibration.

Pump repeatability

Linearity and precision results

Analyte
PIO

Mean RT
3.159  0.001

%RSD for RT
0.03

MET

3.076  0.001

0.04

PIO
168
557125.7
36
0.02

MET
60
455491.2

Nominal conc.
(ng/mL)
24.0
51.0
106.3
221.4
461.2
960.8

Mean calculated conc.

(ng/mL)
23.67  0.35
52.33  1.23
105.73  0.85
225.23  2.63
462.67  12.17
958.07  15.16

33
0.01

2001.6
4003.3

1999.63  39.62
3877.57  110.05
Slope
Intercept

Injection 1
Injection 2
Injection 3

0.000361
0.000369
0.000663

Carryover test
Response in BLK1
Mean peak area response
in AQMIX
Response in BLK2
% Carryover

System suitability
Mean area ratio (PIO/MET)
1.223  0.017

%RSD for area ratio

1.39

direct weighing ensured that the injection volume aspirated

by the autosampler was accurate and precise.
System suitability accounted for the carryover, pump
repeatability and overall system verification. Both carryover
blanks (BLK1 and BLK2) found to be free of any significant
interference at the RT of PIO or MET (5% of the mean peak
area response of PIO (or MET) in AQMIX) (Fig. 1) and the
carryover found to be 0.02% for PIO and 0.01% for MET. %
RSD for retention times of PIO and MET in 6 AQMIX injections
were 0.03% and 0.04%, respectively, which suggested that both
pumps were producing a uniform and constant flow rate. For
overall system verification, peak area response ratio of PIO to
MET was calculated for 6 AQMIX injections and %RSD found to
be 1.39% which suggested HPLC is performing with acceptable
variations using mass spectrometer as detector. The results
for pump repeatability, carryover and system suitability are
summarized in Table 3.
The linearity and precision were performed by plotting
calibration curve of PIO between eight concentration levels,
i.e. 24.00, 51.00, 106.30, 221.40, 461.20, 960.80, 2001.60 and
4003.30 ng/ml and their respective area response ratios to
MET. The precision was calculated at each concentration level
for the three calibration curves and found to be ranging between 0.80 and 2.84% (Table 3). The results indicated that data
generated for linearity and precision for overall system verification was precise, accurate and reproducible.

4.

Conclusion

In the proposed research work, the efforts have been made to

elevate the standards of bioanalytical industry practices for
HPLC calibration and to relieve the actual burden of the process
by considerably reducing the time and resource expenditures
involved. The methodologies have simple procedures and formulas, well defined acceptance criteria as per industry norms
and accurate and precise demonstration of calibration experiments for individual modules and overall system. Moreover, as
the procedure for calibration utilized mass spectrometer software hence undermined the necessity of any extra HPLC

0.000307
0.00114
0.00145

%RSD
1.48
2.36
0.80
1.17
2.63
1.58
1.98
2.84
Coefficient of
correlation
0.9995
0.9999
0.9994

software, its purchase and validation cost. Any software

compatibility issue with the mass spectrometer software is also
out of question. Additionally, calibration of HPLC serves as the
cross-calibration of mass spectrometer as well.

Conflicts of interest
All authors have none to declare.

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