Enhanced Sensitivity Method for MeasuringGemcitabine in Human Plasma

Amit Khatri1, James E. Fisher1, Mark N. Kirstein1,2,&

1 Department of Experimental and Clinical Pharmacology, Clinical Pharmacology Analytical Services, College of Pharmacy,Minneapolis, MN, USA

2 Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA; E-Mail: kirst002@umn.edu

Received: 17 June 2010 / Revised: 12 August 2010 / Accepted: 16 August 2010Online publication: 8 September 2010

Abstract

A simple and reliable method for the separation and sensitive quantitation of gemcitabine inhuman plasma was developed. Samples were concentrated and purified with solid phaseextraction. Extracts were then injected onto Spherisorb 4.6 mm 9 150 mm, 3 lm ODS2column, and isocratically eluted with mobile phase containing 11 mM 1-octanesulfonatesodium as ion pairing agent. The assay was validated and intermediate precision (% CVt)was �6.40%. The assay was linear over a concentration range from 0.01 to 50 lM, and thelower limit of quantitation (LLOQ) was 0.01 lM. Solid phase extraction has enabledachievement of sensitivity which is at least 20-fold less than that reported for other LC assays,and is comparable to MS–MS.

Keywords

Column Liquid ChromatographySolid phase extractionPharmacokinetic studyGemcitabine in human plasma

Introduction

Gemcitabine (dFdC, 20,20-difluorodeoxy-

cytidine) is a deoxycytidine analogue

active against a variety of solid tumors.

It is a prodrug, which undergoes intra-

cellular phosphorylation to form the

active di- (dFdCDP) and triphosphate

(dFdCTP) metabolites. Gemcitabine also

undergoes metabolism by plasma and li-

ver cytidine deaminase (CDA) to form

20,20-difluorodeoxyuridine (dFdU)[1].

Several analytical approaches, includ-

ing F-NMR, enzyme linked immunosor-

bance assay (ELISA), liquid chromatog-

raphy and tandem-mass spectrometry

(MS–MS) have been reported for gemcit-

abine concentration measurements [2–5].

To evaluate gemcitabine population

pharmacokinetics, we developed and

validated an LC method (LLOQ 2 lM)

[5]. Gemcitabine elimination clearance

ranges from 200 to 600 L h-1 [6, 7];

therefore, concentrations rapidly fall be-

low the LLOQ. To help preventing model

misspecification, a more sensitive assay

was needed.

Experimental

Chemicals and Reagents

Gemcitabine as analytical standard was

provided by Eli Lilly (Indianapolis, IN,

USA). Internal standard, 20-deoxycyti-

dine (20dC), and perchloric acid 70%

were from Sigma (St. Louis, MO, USA).

Tetrahydrouridine (THU) was from

Calbiochem (San Diego, CA, USA).

Ion-pairing reagent, 1-octanesulfonate

sodium, was from Regis technologies

(Morton Grove, IL, USA). LC grade

acetonitrile and methanol were obtained

from Fisher (Fairlawn, NJ, USA). MCX

Oasis solid phase extraction cartridges,

3 cc, were from Waters (Milford, MA,

USA). Heparinized human plasma was

from Biological Specialties (Colmar, PA,

USA). All other chemicals were of

analytical grade and obtained from

commercial sources.

2010, 72, 1005–1008

DOI: 10.1365/s10337-010-1751-20009-5893/10/11 � 2010 Vieweg+Teubner Verlag | Springer Fachmedien Wiesbaden GmbH

Limited Short Communication Chromatographia 2010, 72, November (No. 9/10) 1005

Preparation of CalibrationStandards

Gemcitabine (1 mg mL-1) and 20dC (50

lg mL-1) stock solutions were accu-

rately prepared in methanol and stored

at -20 �C. Standard calibration solu-

tions were then further diluted with

methanol. At the time of assay, 20 lL of

each solution was added to 500 lL of

heparinized human plasma to obtain the

standard calibration curve at the fol-

lowing concentrations: 0.01, 0.02, 0.1,

0.5, 2, 10, 40 and 50 lM.

Preparation of Quality ControlSamples

The quality control samples were pre-

pared prior to assay validation. To pre-

vent metabolism of gemcitabine by

plasma cytidine deaminase, tetrahydro-

uridine (THU) was added prior to the

addition of gemcitabine (40 lL of

10 mg mL-1 THU in water per 5.0 mL

of plasma). The samples were then

stored at -80 �C. The internal standard,20dC, was added to obtain a final con-

centration of 9 lM. Gemcitabine quality

control concentrations were 0.075, 0.6,

and 5 lM.

Specimen Preparation andSolid-Phase Extraction

Samples were concentrated with solid

phase extraction (SPE). The SPE

cartridges were washed with 2 mL of

methanol and conditioned with 2 mL of

50 mM NaH2PO4 (pH 2.9) buffer. For

the preparation of calibration standards

in blank plasma, 20 lL each of the stan-

dard calibration solutions and 20 lL of

the internal standard stock solution were

added to the 12 9 75 mm centrifuge tube

and evaporated under nitrogen at 37 �Cfor 5 min. 500 lL of blank plasma con-

taining THU (1:125, THU: plasma) was

added to the dried 12 9 75 mm centri-

fuge tubes, vortex mixed, and acidified

with 500 lL of 4% o-phosphoric acid.

Patient plasma samples were processed

similarly with 20 lL of the internal stan-

dard stock solution. Next, the acidified

plasma was transferred to the condi-

tioned SPE cartridges and centrifuged at

759g for 5 min. The cartridges were then

washed with 2 mL of 50 mM NaH2PO4

(pH 2.9) followed by 2 mL of methanol.

Each washing step was followed by cen-

trifugation at 759g for 3 min. Finally,

gemcitabine was eluted with 1 mL of

freshly prepared methanolic NH4OH

(2% v/v). The eluates were evaporated

under nitrogen at 37 �Cand reconstituted

with 50 lL of mobile phase. After vortex

mixing, 25 lL were injected onto the LC

column.

Instrumentation andChromatographic Conditions

The LC system and data capture soft-

ware has been described previously [5].

25 lL of the standard and sample ex-

tracts were injected onto a Waters

Spherisorb 4.6 mm 9 150 mm, 3 lmODS2 column (Waters Corporation,

Milford, MA, USA) with A-102X in-line

filter, 0.5 lM Frit (Chromtech, Apple

Valley, MN, USA). Analytes were iso-

cratically eluted with a mobile phase

of acetonitrile:methanol:50 mM sodium

phosphate buffer 5:5:90 (v/v) containing

11 mM 1-octanesulfonate sodium. The

aqueous buffer was adjusted to pH 2.9

with 85% o-phosphoric acid. The mobile

phase was filtered with 0.22 micron

Nylon filter (GE Water and Process

Technology, USA). The flow rate was

1.2 mL min-1 and the column tempera-

ture was 40 �C. The wavelength was

267 nm as described previously [5, 8].

Assay Validation

Peak height was used for analysis of gem-

citabine in plasma samples, as described

previously [5]. The linear regression of the

ratio of peakheight of gemcitabine topeak

height of the internal standard (20dC) with

nominal concentrations of gemcitabine in

the samples was weighted by 1/x. The lin-

earity of the calibration curve was evalu-

ated by the squared correlation coefficient.

The procedure was validated over 5 days

by analysis of quality control samples in

quintuplicate to determine the within-day

(repeatability) and total variability (inter-

mediate precision) along with assay accu-

racy and precision [9]. A single set of

calibrators were used for each validation

run.

The lower limit of quantitation

(LLOQ) was defined as the peak height

ratio of signal/noise �10. Gemcitabine

concentrations of 0.01 and 0.02 lMwere

tested in quintuplicate for 5 days during

the validation for determination of

LLOQ. The LLOQ was defined as pre-

cision and accuracy of �10%. Accuracy

parameters were determined with analy-

sis of variance (ANOVA). Percentage

recovery following solid phase extraction

was determined as follows:

Percent recovery

¼ Peak height ratio of extracted plasma standard

Peak height ratio of non - treated standard

� 100:

Results and Discussion

Sample Preparation

During method development, we found

that a protein precipitation step before

solid phase extraction does not provide

any additional benefit. We found that

washing with 2 mL of methanol is suffi-

cient for washing the SPE column, and

1 mL of methanolic NH4OH (2% v/v)

was sufficient for the elution step. This

solid phase extraction enabled us to

concentrate the samples and to achieve

an LLOQ (0.01 lM), which is at least 20

fold less than the previously reported

values from LC-UV methods and com-

parable to a 0.01–0.04 lM LLOQ range

in methods [2, 10, 11].

Separation

Gemcitabine was separated from endoge-

nous plasma components using a Waters

Spherisorb 4.6 mm 9 250 mm, 5 lmODS2 column with isocratic elution.

We used reversed-phase chromatography

with a mobile phase at pH 2.9 and

11.0 mM 1-octanesulfonate sodium as

an ion-pairing reagent for the best

results. We initially started with 3 mM

1006 Chromatographia 2010, 72, November (No. 9/10) Limited Short Communication

1-octanesulfonate sodium as described

previously [5]; however, further adjustment

to 11 mM was required to achieve better

separation from an interfering endogenous

substance in the plasma. Due to the higher

concentration of ion-pairing agent used

and the presence of a late eluting endoge-

nous substance in the plasma, the run

time was about 25 min on the 250 mm

column. Hence further method develop-

ment and validation was performed on a

4.6 mm 9 150 mm, 3 lm ODS2 column,

which resulted in shorteningof the run time

to 16 min. Acetonitrile 5% (test range

4–12%) and methanol 5% (test range

4–12%) in the mobile phase with a column

temperature of 40 �C produced the best

resolution from endogenous substances.

The retention times for 20dC and gemcita-

bine were 6.8 and 9.5 min, respectively, at

which no interfering endogenous peaks

were detected in human plasma.

Assay Validation

Figure 1 shows representative chro-

matogram overlays of increasing gemcit-

abine concentrations with internal

standard in human plasma. Accuracy and

precision data for gemcitabine calibra-

tion standards are shown in Table 1.

Accuracy of the calibration standards

ranged from 96 to 108% and precision,

from 1 to 7%. The mean (±SD) weighted

linear regression slope, intercept, and

coefficients of determination (r2 value)

were as follows: slope = 0.087994 ±

0.0048, intercept = -0.00014 ± 0.00025,

and r2 = 0.9998 ± 0.00017, respectively.

Assessment of within-day and total vari-

ability in assay performance, precision,

and accuracy was performed by an anal-

ysis of variance procedure (Table 1). The

lowest concentration of the calibration

curve, 0.01 lM, had an accuracy and

precision of 102 and 7.2%, respectively;

hence assessed to be the LLOQ of the

assay. The calibration curve was linear up

to 50 lM. The average recoveries for

gemcitabine and internal standard were

62.63 and 82.68%, respectively.

Patient Samples

Application of this assay enabled us to

measure additional patient plasma sam-

ples that were below a measurable limit

with the previously published assay [5].

The concentration–time data from a

representative patient were modeled in

ADAPT-II, and the clearance was esti-

mated as 420.7 L h-1 [12].

Conclusion

We developed a simple and reliable

method with enhanced sensitivity for the

separation and quantitation of gemcita-

bine in human plasma. The lower limit of

quantitation for gemcitabine in our assay

was 0.01 lM,which is at least 20-fold less

than that reported for other LC-UV

assays, and comparable to those reported

for MS–MS methods.

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150

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Time (min)m

V

Internal standard Gemcitabine

Blank plasma

Fig. 1. Representative chromatogram overlays of human plasma as blank, and with internalstandard and 0.1, 0.5, 2 and 10 lM gemcitabine calibration standards. Inset shows magnifiedchromatogram overlays for the lowest concentration (0.01 lM) and human plasma as blank

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