Paresh Varshney a,*, Atul KauParmesh Kumar Dwivedi d

a th Univ

on Phar

& Deve

Univers

ion of the instrument

.

al modules suggested

g the correct injection

utosampler chambers

e accuracy, precision,

t carryover.

dia Pvt. Ltd. All rights

reserved.

spectrometry (LCeMS/MS) has been the mainstay of bio-

analytical 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

d endogenous com-

pounds in biological matrices.

conventional procedures for HPLC calibration which use

Photodiode Array (PDA) or variable UV/Visible spectropho-

tometers as detectors.8e14 However, due to continuous evo-

lution 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.

Available online at www.sciencedirect.com

.e

i n t e rn a t i o n a l j o u rn a l o f c h em i c a l a n d an a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 1 0 2e1 0 7E-mail address: varshneyparesh@gmail.com (P. Varshney).High-performance liquid chromatography coupled to mass The bioanalytical laboratories throughout theworld rely on1. Introduction small molecule drugs, metabolites an4e7HPLC calibration

LCeMS/MS

Injection volume repeatability test

Injector position check

System suitability

and precision were carried out for holistic (overall system) calibrat

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

Results/Conclusion: The results of calibration experiments for individu

that pumps are yielding accurate flow rate, autosampler is aspiratin

volume irrespective to position or volume, and column oven and a

are maintaining steady temperatures. Holistic calibration verified th

repeatability and reproducibility of the instrument with insignifican

Copyright 2013, JPR Solutions; Published by Reed Elsevier InKeywords: illustrated using direct weighing method. Carryover test, pump repeatability test, linearityDepartment of Pharmacology, Sai Nab Formulation and Development, WatscAnalytical Division, Dabur ResearchdAmity Institute of Pharmacy, Amity

Lucknow 226010, Uttar Pradesh, India

a r t i c l e i n f o

Article history:

Received 25 February 2013

Accepted 2 April 2013

Available online 9 April 20130976-1209/$ e see front matter Copyright http://dx.doi.org/10.1016/j.ijcas.2013.04.001shik b, Alok Sharma c, Sajal Srivastava d,

ersity, Ranchi, Jharkhand 835217, India

ma Pvt Ltd., Mumbai 400099, India

lopment Centre, Dabur India Ltd., Sahibabad 201010, India

ity Uttar Pradesh, Lucknow Campus Gomti Nagar Extension,

a b s t r a c t

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

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

which simulates the routine analysis in bioanalytical laboratories.

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 weremass spectrometer as detector

A rational approach for HPLC calibration usingOriginal Article

journal homepage: www2013, JPR Solutions; Publilsevier .com/locate/ i jcasshed by Reed Elsevier India Pvt. Ltd. All rights reserved.

observed for the number of FDA 483 observations for

HPLC calibration while using hyphenated LCeMS/MS in-

struments.15,16 The limitations to the conventional ap-

proaches for HPLC calibration are summarized below.

The assessment of injector position check (IPC) and in-

jection volume repeatability test (IVRT) is done by statistical

comparison between peak area response and the concen-

tration 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 interpreta-

sensitivity UV detector for holistic (overall) calibration. As

bioanalytical methods involve analysis of drugs at very low

2.1. 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) andmethanol was sourced fromS.D. fine chem

limited (Mumbai, India). HPLC grade water was obtained from

aMilli-Q gradient A10 purifier. Ammonium formate (analytical

grade) was sourced from Panreac (Barcelona, Spain).

i n t e r n a t i o n a l j o u r n a l o f c h em 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 2e1 0 7 103concentrations, it is essential to ensure that even the slight

variations in the performance of HPLC are noticed by the de-

tector 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 in-

process evidence documents are generated.

The proposed research work is aimed to demonstrate

methodologies for the calibration of HPLC using highly sen-

sitive mass spectrometer as detector to overcome the limita-

tions 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 cali-

bration was performed coupling HPLC in-tandem with mass

spectrometer.

2. 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 vali-

dated over the concentration range of 24.00e4003.30 ng/ml for

PIO using Metaxalone (MET) as an Internal Standard (IS).

Table 1 e Optimized parameters for mass spectrometry.

Source and gas parameters

Curtain gas (psi) 25

Ion spray voltage (volts) 5000

Heater gas (psi) 50

Source temperature (C) 550Nebulizer gas (psi) 50tion. However, the procedures count on the indirect mea-

surement 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 compro-

mised due to unnoticed hardware alterations or aging

components.

Second limitation is the irrational coupling of HPLC to lowCollision associated dissociation gas (psi) 6Compound parameters

PIO MET

Declustering potential (volts) 80 32

Entrance potential (volts) 5 10

Collision energy (volts) 25 14

Collision cell exit potential (volts) 3 32.4. 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.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 onWaters XTerraMS

C18 5 mm (4.6mm 150mm) column at a constant flow rate of0.5 ml/min. The temperatures of the column oven and auto-

sampler 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 positivemode 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 1.2.2. Instrumentation

Holistic calibration was carried out on Perkin Elmer HPLC (se-

ries 200 LC pumps, series 200 column oven, series 200 auto-

sampler) connected in-tandem with calibrated API-3200 triple

quadrupole mass spectrometer (from MDS Sciex, Applied Bio-

systems, Canada). Gas generator (from Peak Scientific, Scot-

land), Model NM20ZAwas used to supply nitrogen gas and zero

grade air. The instrumentation was monitored using Analyst

software version 1.5 for data acquisition and processing.Dwell time (msec) 200 200

ference between the mean of actual temperature and set

Global %RSD was also calculated for the injection volume. %

pump repeatability and overall system verification using

UV detector is a time consuming procedure and typically take

i n t e rn a t i o n a l j o u rn a l o f c h em i c a l a n d an a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 1 0 2e1 0 7104RSD should be

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) % Deviation

Pump 1 Pump 2

0.2 0.200954 0.48 0.201691 0.85

0.5 0.50324 0.65 0.504815 0.96

1.0 1.005985 0.60 0.991259 0.872.0 1.983424 0.83 1.988246 0.59

IPC Results IVRT Results

Vial Position Mean injectionvolume (mL)

%RSD Set injectionvolume (mL)

Actual injectionvolume (mL)

Injection volumeaccuracy

1 9.88 0.04 0.44 5 5.01 100.2010 9.98 0.11 1.06 10 10.02 100.1755 9.95 0.03 0.25 15 15.05 100.3691 10.00 0.11 1.05 20 20.00 99.98100 9.96 0.09 0.86 25 24.97 99.89Mean global (mL) 9.95 0.08 Mean injection volume accuracy 100.12 0.18%RSD global 0.80 Coefficient of correlation (R2) 1

i n t e r n a t i o n a l j o u r n a l o f c h em 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 2e1 0 7 105laboratory, the holistic calibration of HPLCwas completed in a

day using in-tandemmass spectrometer as detector with a set

of meaningful acceptance criteria that conform to the in-

dustry 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 mar-

gins of error as high sensitivity analytical balance was used to

mark the volume measurement. The process was time effi-

cient. 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 was0.00e0.03 C from the set temperatures. Datalogger was used

Fig. 1 e Representative LCeMS/MS chromatograms of BLK1to 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 be-

tween 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. Theprecision for IVRT was evaluated in terms of %RSD for injec-

tion volume accuracy between different injection levels and

found to be 0.18 (Table 2). As IPC and IVRT were executedwithout any detector, completed with ease in few hours, and

, AQMIX and BLK2 of PIO and MET for carryover test.

Conflicts of interest

r e f e r e n c e s

Mean peak area response

in AQMIX

557125.7 455491.2

i n t e rn a t i o n a l j o u rn a l o f c h em i c a l a n d an a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 1 0 2e1 0 7106direct 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 peakarea 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

METwas 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

Response in BLK2 36 33

% Carryover 0.02 0.01

System suitability

Mean area ratio (PIO/MET) %RSD for area ratio

1.223 0.017 1.39Table 3 e Results for holistic calibration.

Pump repeatability

Analyte Mean RT %RSD for RT

PIO 3.159 0.001 0.03

MET 3.076 0.001 0.04

Carryover test

PIO MET

Response in BLK1 168 60calibration 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 be-

tween 0.80 and 2.84% (Table 3). The results indicated that data

generated for linearity and precision for overall system veri-

fication 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 for-

mulas, well defined acceptance criteria as per industry norms

and accurate and precise demonstration of calibration experi-

ments for individual modules and overall system. Moreover, as

the procedure for calibration utilized mass spectrometer soft-

ware hence undermined the necessity of any extra HPLC1. Xu RN, Fan L, Rieser MJ, El-Shourbagy TA. Recent advances inhigh-throughput quantitative bioanalytical analysis byLCeMS/MS. J Pharm Biomed Anal. 2007;44:342e355.All authors have none to declare.software, its purchase and validation cost. Any software

compatibility issuewith themass spectrometer software is also

out of question. Additionally, calibration of HPLC serves as the

cross-calibration of mass spectrometer as well.

Linearity and precision results

Nominal conc.

(ng/mL)

Mean calculated conc.

(ng/mL)

%RSD

24.0 23.67 0.35 1.4851.0 52.33 1.23 2.36106.3 105.73 0.85 0.80221.4 225.23 2.63 1.17461.2 462.67 12.17 2.63960.8 958.07 15.16 1.58

2001.6 1999.63 39.62 1.984003.3 3877.57 110.05 2.84

Slope Intercept Coefficient of

correlation

Injection 1 0.000361 0.000307 0.9995

Injection 2 0.000369 0.00114 0.9999

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i n t e r n a t i o n a l j o u r n a l o f c h em 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 2e1 0 7 107

A rational approach for HPLC calibration using mass spectrometer as detector1. Introduction2. Materials and methods2.1. Chemicals and reagents2.2. Instrumentation2.3. LCMS/MS conditions during holistic calibration2.4. Calibration parameters2.5. PFRA test2.6. COTP and ASTP check2.7. Injector position check2.8. Injection volume repeatability test2.9. Holistic calibration

3. Results and discussion4. ConclusionConflicts of interestReferences