research article high performance liquid chromatographic...

Research ArticleHigh Performance Liquid Chromatographic Assay forthe Simultaneous Determination of Posaconazole andVincristine in Rat Plasma

Hadeel A Khalil1 Ahmed F El-Yazbi2 Tarek S Belal1 and Dalia A Hamdy1

1Pharmaceutical Analytical Chemistry Department Faculty of Pharmacy Alexandria University Alexandria 21521 Egypt2Department of Pharmacology Faculty of Pharmacy Alexandria University Alexandria 21521 Egypt

Correspondence should be addressed to Dalia A Hamdy drdaliahamdygmailcom

Received 27 October 2015 Revised 28 November 2015 Accepted 2 December 2015

Academic Editor David M Lubman

Copyright copy 2015 Hadeel A Khalil et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Purpose Developing a validated HPLC-DAD method for simultaneous determination of posaconazole (PSZ) and vincristine(VCR) in rat plasma Methods PSZ VCR and itraconazole (ITZ) were extracted from 200120583L plasma using diethyl ether in thepresence of 01M sodium hydroxide solution The organic layer was evaporated in vacuo and dried residue was reconstituted andinjected through HC-C18 (46 times 250mm 5120583m) column In the mobile phase acetonitrile and 0015M potassium dihydrogenorthophosphate (30 70 to 80 20 linear gradient over 7 minutes) pumped at 15mLmin VCR and PSZ were measured at 220 and262 nm respectively Two Sprague Dawley rats were orally dosed PSZ followed by iv dosing of VCR and serial blood sampling wasperformed Results VCR PSZ and ITZ were successfully separated within 11min Calibration curves were linear over the rangeof 50ndash5000 ngmL for both drugs The CV and error of the mean were le18 and limit of quantitation was 50 ngmL for bothdrugs Rat plasma concentrations of PSZ and VCRwere simultaneously measured up to 72 h and their calculated pharmacokineticsparameters were comparable to the literature Conclusion The assay was validated as per ICH guidelines and is appropriate forpharmacokinetics drug-drug interaction studies

1 Introduction

Vincristine (VCR) is a mitotic inhibitor antineoplastic agentthat is used mainly in combination chemotherapy regi-mens for acute and chronic leukemia lymphomas includ-ing Hodgkinrsquos disease and non-Hodgkinrsquos lymphomas andmultiple myeloma [1] Posaconazole (PSZ) is an oral triazoleantifungal used in the treatment of severe oropharyngealcandidiasis invasive aspergillosis and other fungal infectionsin patients who are resistant to or intolerant of otherantifungal drugs PSZ is also given for prophylaxis in patientswho are at high risk for invasive fungal disease due toimmunosuppression such as haematopoietic stem cell trans-plant recipients with graft-versus-host disease or those withhaematological malignancies with prolonged neutropenia asa result of chemotherapy [1 2]The coadministration of azoles(as prophylaxis or treatment of fungal infections) and VCRhas been shown to increase VCR neurotoxic effects due to

the inhibition of cytochrome P450 (CYP) isoform 3A4 forwhich VCR is a substrate [2] Those neurotoxic symptomsusually appear as constipation and peripheral neurotoxicity[3] In addition few case reports have illustrated the possibleexacerbation of VCR toxicity by coadministration of PSZin children and young adults where fluctuations in level ofconsciousness and seizures have been observed [4]

PSZhas been determined in humanplasma samples usingcapillary electrophoresis [5] HPLC with fluorescence detec-tion [6] and HPLC-tandem mass spectrometry (HPLC-MS-MS) methods [7 8] Additionally the simultaneous determi-nation of azole antifungals including PSZ in plasmaserumsamples gained a growing interest in the past few yearsDifferent chromatographic methods have been describedfor this task such as micellar electrokinetic chromatography(MEKC) [9] and several liquid chromatographic methodscoupledwith tandemmass spectrometry [10 11] fluorescencedetection [12] and UV detection [13ndash16] On the other

Hindawi Publishing CorporationInternational Journal of Analytical ChemistryVolume 2015 Article ID 743915 6 pageshttpdxdoiorg1011552015743915

2 International Journal of Analytical Chemistry

hand estimation of VCR in plasma samples has been carriedout using HPLC-UV detection [17 18] UPLC-MS-MS [19]and several HPLC-MS-MS methods [20ndash23] Besides anHPLC-MS-MS method was presented for the simultaneousdetermination of VCR and actinomycin-D in human driedblood spots [24] In addition quantification of intracellularVCR concentrations in children with acute lymphoblasticleukemia has been described using HPLC with electro-chemical detection procedure [25] Moreover capillary zoneelectrophoresis coupled with electrochemical detection hasbeen employed to study the uptake kinetics of VCR by humanerythrocytes [26] Finally pharmacokinetics of PSZ and VCRin rats have been separately discussed in only two publishedreports [27 28]

Although PSZ-VCR drug-drug interaction has beenexposed in several previous reports the concurrent deter-mination of PSZ and VCR has not been tackled yet sinceno analytical reports can be found in the literature In thisreport we describe the first simple and reliable RP-HPLCwith diode array detection procedure for the simultaneousdetermination of PSZ andVCR in rat plasmaThe structurallyrelated azole antifungal drug (itraconazole ITZ) has beenused as an internal standard The proposed method has beenapplied in a preliminary pharmacokinetics drug interactionstudy in rats An analytical method capable of simultaneouslymeasuring two drugs would be a valuable tool facilitatingthe measurement of pharmacokinetics using a single blooddraw for each time point with no need to split the samplefor processing through two assay methods In serial bloodcollection studies involving rats in particular this is animportant consideration due to the need to keep cumulativeblood volume withdrawal to a minimum

2 Experimental

21 Materials and Reagents Posaconazole and vincristinesulfate powders (purity gt99 for both) were purchasedfrom Selleckchem (Houston TX USA) Itraconazole wasa kind gift from Nifty Labs Pvt Ltd Hyderabad IndiaHPLC grade methanol and acetonitrile (Fisher ScientificUK Limited Loughborough Leicestershire UK) analyti-cal grade potassium dihydrogen orthophosphate (Riedel-de-Haen Germany) and high purity distilled water wereused Noxafil oral suspension labeled to contain 40mgmLPSZ (Patheon Inc Ontario Canada) was purchased fromSchering-Plough SA Vincarine vials labeled to contain1mgmL vincristine sulfate solution (EIMC United Pharma-ceuticals Badr city Cairo Egypt) were obtained from thelocal market

22 Chromatographic Conditions The HPLC-DAD systemconsisted of Agilent 1200 series (autoinjector quaternarypump vacuum degasser and diode array and multiplewavelength detectors G1315 CD and G1365 CD) connectedto a computer loaded with Agilent ChemStation Software(Agilent Technologies Santa Clara CA USA) The DADwavelength was set at 220 and 262 nmThe chromatographicseparationwas achieved using aHC-C18 (46times 250mm 5120583m

particle size) column attached to HC-C18 (46 times 125mm5 120583m particle size) guard column (Agilent TechnologiesSanta Clara CA USA) The gradient elution composed themobile phase acetonitrile and 0015Mpotassiumdihydrogenorthophosphate (30 70 to 80 20 linear over 7 minutes)pumped at 15mLmin Injection volume was 80120583L Alldeterminations were performed at 25∘C

23 Stock and Standard Solutions Stock solutions of PSZand VCR sulphate (100mgL) were separately prepared inmethanol A 10mgL stock solution of the internal standard(ITZ) was prepared in methanol To prepare samples for thecalibration curves and validation assessment three workingstandard solutions (10 1 and 01mgL) of PSZ and VCRwere prepared freshly by successive 110 dilutions of the stocksolutions with methanol

24 Extraction Procedure The ITZ (003mL) was added toeach 02mL rat plasma sample in a glass test tube To therat plasma sample 003mL of 01M NaOH and 6mL ofdiethyl ether were added Because methanol was present inthe standard curve samples an equivalent amount was addedto the test samples as well (02mL) The tubes were coveredvortex-mixed for 2min at high speed and then subsequentlycentrifuged for 10min at sim2500timesg The organic layer wastransferred to new glass tubes and evaporated to drynessin vacuo (Christ rotational vacuum concentrator Germany)The tubes were placed in a minus20∘C freezer until analysis timeThe residues were reconstituted in 100 120583L methanol of which80 120583L volumes were injected into the HPLC

25 Recovery The plasma recoveries were determined forPSZ VCR and ITZ at concentration level 2500 ngmL inrat plasma using four replicates for each concentration Theextraction efficiency was determined by comparing the peakareas of each analyte to the peak areas of the same amountsdirectly injected to the instrument without extraction

26 Validation Calibration curves were constructed usingsamples of 02mL rat plasma containing PSZ VCR and ISThe curve ranged from 50 to 5000 ngmL for both PSZ andVCR The ratios of PSZ and VCR peak areas to IS peak areawere calculated and plotted versus the expected PSZ or VCRconcentrations Owing to the wide range of concentrationsthe calibration curve data were weighed by a factor of 11199092for both drugs Intraday accuracy and precision of the assaywere determined using four sample replicates of 50 500and 2500 ngmL for each of PSZ and VCR in rat plasmain the same day The same concentrations were analyzed inthree separate days for assessment of the interday accuracyand precision For each daily run concentrations weredetermined by comparison with a calibration curve preparedsimultaneously on the same day of analysis Precision wasdetermined using percentage coefficient of variation (CV)and bias was assessed using percentage error of the mean

27 Application to a Drug Interaction PK Study To evaluatethe applicability of this method in vivo two rats (200ndash250 g)

International Journal of Analytical Chemistry 3

were given 40mgkg PSZ orally followed by 01mgkg VCRiv through the tail vein after 30min of oral dosing Theprotocol was approved by the Ethics Committee Facultyof Pharmacy Alexandria University On the day before thepharmacokinetic study food was withheld overnight On thenext morning animals were transferred to metabolic cagesto conduct the pharmacokinetic experiments Serial bloodsamples were collected at 050 075 092 133 20 35 60 80240 480 and 720 h after oral PSZ dose using retroorbitalsampling Plasma was separated by centrifugation of theblood at sim2500timesg for 3minThe samples were kept at minus20∘Cuntil analysis time

28 Data and Statistical Analysis Noncompartmental meth-ods were applied to calculate the pharmacokinetic param-eters The elimination rate constant (120582119911) was calculated bysubjecting the plasma concentrations in the terminal phaseto linear regression analysis 119905(12) was calculated using theequation 119905(12) = 0693120582119911 AUC

0ndashinfin was calculated usingthe combined log-linear trapezoidal rule from time 0 h afterdose to the time of the last measured concentration plusthe quotient of the last measured concentration divided by120582119911 The concentration at time 0 h after iv dosing (119862o) wasestimated by back extrapolation of the log-linear regressionline using the first three measured plasma concentrations totime 0 The clearance was calculated as the quotient of doseto AUC

0ndashinfin All compiled data were reported as mean plusmn SDunless otherwise indicated

3 Results

VCR PSZ and ITZ eluted at retention times 59 83 and104min respectively (Figure 1) The total analysis run timewas sim11min The three peaks were almost symmetricalwith excellent baseline separation and no interferences fromendogenous substances in plasma The column separationfactor (120572) and resolution factor for VCR and PSZ werecalculated to be 158 and 145 for VCR and 132 and 795for PSZ respectively The column capacity factors (1198701015840) forVCR PSZ and ITZ were calculated to be 243 383 and 505respectively

The VCR extraction efficiency recovery was found to be757 plusmn 54 while PSZ and ITZ showed 1039 plusmn 31 and1036plusmn54 (sim100) recovery respectivelyThe assay linear-ity for VCR and PSZ calculated by measuring the peak arearatios of analyte internal standard within the concentrationrange of 50 to 5000 ngmL showed high linearity with amean1199032

ge 0997 for both drugs (Figure 2) The mean weightedslope and intercept were found to be 00007plusmn509times10minus5 and0315 plusmn 0375 for PSZ respectively and 00004 plusmn 00002 andminus0002 plusmn 00037 for VCR respectively

The validation data showed the assay to be sensitiveaccurate and precise with the intraday and interday CVvalues less than or equal to 132 and 878 for both drugsrespectively (Table 1) The mean interday error was less than131 for both drugs Since both CV of interday andintraday assessment and interday mean error yielded valuesless than 20 at the lowest concentration tested the lower

VCRITZ

2 4 6 8 10 12 140(min)

0

10

20

30

40

60

50

(mAU

)

PSZ VCR

(mAU

)

04080

120

2 3 4 61 5

(min)

(a)

2 4 6 8 10 12 140(min)

minus100

102030405060

(mAU

)

(b)

minus50minus25

0255075

100125150175190

(mAU

)

2 4 6 8 10 120(min)

(c)

Figure 1 Chromatogram of (a) rat plasma spiked with 2500 ngmLofVCR andPSZ (b) rat plasma obtained after 6 h of oral PSZ dosingand (c) blank rat plasma measured at 262 nm The inset shows asection of a chromatogram for rat plasma spiked with 2500 ngmLof VCR measured at 220 nm

limit of quantitation (LLQ) based on 02mL of rat plasmawas50 ngmL for both drugs

In both rats dosed with 40mgKg PSZ orally followedby 01mgKg VCR iv 05 h later the PSZ showed higherplasma concentrations thanVCR at all time points (Figure 3)The PSZ Cmax for the two rats were 2344 and 1056 ngmLand the VCR 119862o were 376 and 228 ngmL respectively ThePSZ AUC

0ndashinfin were 206459 and 67744 ngsdothmL whereas theVCR AUC

0ndashinfin were 2347 and 2568 ngsdothmL for rat 1 andrat 2 respectively The PSZ tmax was found to be 84 and78 h for both rats 1 and 2 respectively The VCR clearanceand half-life were calculated to be 0043 LhKg and 192 hfor rat 1 and 0038 LhKg and 148 h for rat 2 respectively


Table 1 Precision and accuracy for the determination of PSZ and VCR in rat plasma using the proposed HPLC-DADmethod

NominalconcentrationngmL

Drug Intraday mean plusmn SD (intraday CV) Interday mean plusmnSD ngmL

InterdayCV

Interdaymean error

50 PSZ 573 plusmn 105 (182) 549 plusmn 954 (174) 574 plusmn 086 (149) 565 plusmn 005 136 131VCR 503 plusmn 599 (119) 562 plusmn 221 (393) 473 plusmn 307 (648) 513 plusmn 45 878 253


2500 PSZ 2460 plusmn 383 (156) 2413 plusmn 127 (525) 2579 plusmn 270 (105) 2483 plusmn 857 345 minus065VCR 2494 plusmn 282 (113) 2646 plusmn 186 (703) 2476 plusmn 264 (107) 2539 plusmn 936 369 154

PSZ VCR

R2 = 09982

y = 00007x + 01884

005

115

225

335

4

Peak

area

ratio

1000 2000 3000 4000 5000 60000Concentration (ngmL)

R2 = 09966

y = 00006x minus 00581

0

05

1

15

2

25

3

Peak

area

ratio


Figure 2 Linearity and regression for the determination of PSZ and VCR in rat plasma

1

10

100

1000

10000

Con

cent

ratio

n (n

gm

L)

0 20 40 60Time (h)

VCRPSZ

VCRPSZ

1

10

100

1000

10000

0 20 40 60

Con

cent

ratio

n (n

gm

L)

Time (h)

Figure 3 Plasma concentration versus time curves for PSZ and VCR in two rats that were given 40mgkg PSZ orally followed by iv dosingof 01mgkg VCR 30 minutes later

The assay managed to measure the plasma concentrations forboth drugs up to 72 h after dose

4 Discussion

This paper demonstrates an easy rapid sensitive HPLC assayfor the simultaneous determination of VCR and PSZ in ratplasma It successfully measures their plasma concentrationsand calculates their PK parameters up to 72 h after dose Itis worth mentioning that the assay was applied by 3 different

analysts on two different HPLC apparatus and yielded similarresults with minor changes in retention times and totalruntimeThe assay was also successfully applied to determineboth drugsrsquo concentrations in other matrices ratrsquos liver tissuehomogenate hyperlipidemic ratrsquos plasma and liver tissuehomogenate in addition to Sorenson buffer (pH = 74) withexcellent linearity and same limit of quantification (data notshown)

During assay optimization the authors utilized the exactchromatographic conditions that they recently published for


the HPLC determination of PSZ in bulk form and suspensiondosage form [29] However HC-C18 instead of Zorbax SB-C18 column was utilized and HC-C18 guard column wasadded Despite the fact that VCR was detected at 262 nm(Figure 1) it showed better sensitivity and yield at the shortwavelength 220 nmThis demonstrates the importance of themultiple wavelength detector (DAD) which offers the advan-tage of measuring each analyte at its optimum wavelengththus improving sensitivity

During the optimization of the extraction procedures theauthors tried the direct protein precipitationmethod and liq-uid extraction using diethyl ether ethyl acetate or methylenechloride Both direct protein precipitation and diethyl etherextraction procedures showed similar recoveries Howeverthe extraction procedure was preferred to protect the columnmaterials and for the ease of the evaporation step in vacuocompared to the direct protein precipitation one Solid phaseextraction was not attempted due to its relatively higher costwhenworkingwith large number of sampleswithin the futureplanned PK studies

ThePSZ andVCR retention times using our simultaneousassay lie within the reported retention times for PSZ andVCR respectively [13ndash18] There is a slight interference froman endogenous plasma peak in the PSZpeak this interferenceresulted in the fact that we could not quantify the PSZpeak accurately below the 50 ngmL concentration howeveras mentioned before it did not interfere with any of thevalidation parameters within our concentration range 50ndash5000 ngmL

The pharmacokinetics parameters reported for PSZand VCR in rat were compared to the only two similarmanuscripts found in literature [30 31] The VCR clearancecame within the lower border of that reporting 012 plusmn008 LhKg and showed similar terminal phase half-life tothat reporting 143plusmn63 h [30] Similarly PSZmeanAUC and119862max were close to those reported previously [31] The slightvariability could be due to the concomitant administrationwith VCR or difference between our SD rats and the Crl CDBR reported [32] It is worth mentioning that for the futureplanned elaborated VCR-PSZ drug interaction study thedesign of the drug interaction needs to be modified to mimicthe actual conditions occurring in humans However forthe purpose of proving the appropriateness of the analyticaltechnique to study such interaction we successfully dosedonly a single dose of each drug and managed to measure itusing our DAD detector for up to 72 h which is advantageousover similar HPLC-DAD assays reported for each drug alone

5 Conclusion

This paper describes the first validated method for the simul-taneous determination of posaconazole and vincristine in ratplasmaThe proposed HPLC-DADmethod has been appliedin the investigation of their pharmacokinetics in rats up to72 h after dose The obtained pharmacokinetics parametersfor posaconazole and vincristine are in good agreement withthose reported in previous reports The developed method issimple sensitive and suitable for the estimation of both drugs

in comprehensive drug-drug interaction pharmacokineticstudies in rats

Conflict of Interests

The authors declare no financial personal or organizationalconflict of interests

Acknowledgment

The studywas funded by Science ampTechnologyDevelopmentFund (STDF) (Project ID 5575) Ministry of State for Scien-tific Research Egypt

References

[1] S C Sweetman EdMartindalemdashTheComplete Drug ReferenceThe Pharmaceutical Press London UK 36th edition 2009

[2] D A Hamdy S El-Salem H El-Geed and M Zaidan ldquoPosa-conazole a prophylactic therapy in patients with haematolog-ical cancer drug use evaluation studyrdquo European Journal ofHospital Pharmacy Science and Practice vol 20 no 4 pp 223ndash226 2013

[3] RM van Schie R J M Bruggemann PM Hoogerbrugge andD M W M te Loo ldquoEffect of azole antifungal therapy on vin-cristine toxicity in childhood acute lymphoblastic leukaemiardquoJournal of Antimicrobial Chemotherapy vol 66 no 8 pp 1853ndash1856 2011

[4] D A Hamdy H El-Geed S El-Salem and M Zaidan ldquoPosa-conazole-vincristine coadministration triggers seizure in ayoung female adult a case reportrdquo Case Reports in Hematologyvol 2012 Article ID 343742 3 pages 2012

[5] H-W Liao S-W Lin U-I Wu and C-H Kuo ldquoRapid andsensitive determination of posaconazole in patient plasma bycapillary electrophoresis with field-amplified sample stackingrdquoJournal of Chromatography A vol 1226 pp 48ndash54 2012

[6] W Neubauer A Konig R Bolek et al ldquoDetermination ofthe antifungal agent posaconazole in human serum by HPLCwith parallel column-switching techniquerdquo Journal of Chro-matography B Analytical Technologies in the Biomedical and LifeSciences vol 877 no 24 pp 2493ndash2498 2009

[7] J X Shen G Krishna and R N Hayes ldquoA sensitive liq-uid chromatography and mass spectrometry method for thedetermination of posaconazole in human plasmardquo Journal ofPharmaceutical and Biomedical Analysis vol 43 no 1 pp 228ndash236 2007

[8] J M Cunliffe C F Noren R N Hayes R P Clement and J XShen ldquoA high-throughput LC-MSMS method for the quanti-tation of posaconazole in human plasma implementing fusedcore silica liquid chromatographyrdquo Journal of Pharmaceuticaland Biomedical Analysis vol 50 no 1 pp 46ndash52 2009

[9] S-C Lin H-Y Liu S-W Lin et al ldquoSimultaneous deter-mination of triazole antifungal drugs in human plasma bysweeping-micellar electrokinetic chromatographyrdquo Analyticaland Bioanalytical Chemistry vol 404 no 1 pp 217ndash228 2012

[10] A Chahbouni A J Wilhelm J C G Den Burger A Sinjeweland RM Vos ldquoValidated liquid chromatography-tandemmassspectroscopy method for the simultaneous quantification offour antimycotic agents in human serumrdquo Therapeutic DrugMonitoring vol 32 no 4 pp 453ndash457 2010


[11] J-F Jourdil J Tonini and F Stanke-Labesque ldquoSimultane-ous quantitation of azole antifungals antibiotics imatiniband raltegravir in human plasma by two-dimensional high-performance liquid chromatography-tandem mass spectrome-tryrdquo Journal of Chromatography B vol 919-920 pp 1ndash9 2013

[12] S L Buckner M M Ceesay A Pagliuca P E Morgan and RJ Flanagan ldquoMeasurement of posaconazole itraconazole andhydroxyitraconazole in plasmaserum by high-performanceliquid chromatography with fluorescence detectionrdquoTherapeu-tic Drug Monitoring vol 33 no 6 pp 735ndash741 2011

[13] S Chhun E Rey A Tran O Lortholary G Pons and V JullienldquoSimultaneous quantification of voriconazole and posaconazolein human plasma by high-performance liquid chromatographywith ultra-violet detectionrdquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol852 no 1-2 pp 223ndash228 2007

[14] J-B Gordien A Pigneux S Vigouroux et al ldquoSimultaneousdetermination of five systemic azoles in plasma by high-performance liquid chromatographywith ultraviolet detectionrdquoJournal of Pharmaceutical and Biomedical Analysis vol 50 no5 pp 932ndash938 2009

[15] M Zhang G A Moore M L Barclay and E J Begg ldquoAsimple high-performance liquid chromatography method forsimultaneous determination of three triazole antifungals inhuman plasmardquo Antimicrobial Agents and Chemotherapy vol57 no 1 pp 484ndash489 2013

[16] C P W G M Verweij-van Wissen D M Burger P E VerweijR E Aarnoutse and R J M Bruggemann ldquoSimultaneousdetermination of the azoles voriconazole posaconazole isavu-conazole itraconazole and its metabolite hydroxy-itraconazolein human plasma by reversed phase ultra-performance liquidchromatography with ultraviolet detectionrdquo Journal of Chro-matography B vol 887-888 pp 79ndash84 2012

[17] L Embree K A Gelmon A W Tolcher et al ldquoValidation ofa high-performance liquid chromatographic assay method forquantification of total vincristine sulfate in human plasma fol-lowing administration of vincristine sulfate liposome injectionrdquoJournal of Pharmaceutical and Biomedical Analysis vol 16 no4 pp 675ndash687 1997

[18] J Chen H He S Li and Q Shen ldquoAn HPLC method forthe pharmacokinetic study of vincristine sulfate-loaded PLGA-PEG nanoparticle formulations after injection to ratsrdquo Journalof Chromatography B vol 879 no 21 pp 1967ndash1972 2011

[19] F Yang H Wang M Liu P Hu and J Jiang ldquoDeterminationof free and total vincristine in human plasma after intravenousadministration of vincristine sulfate liposome injection usingultra-high performance liquid chromatography tandem massspectrometryrdquo Journal of Chromatography A vol 1275 pp 61ndash69 2013

[20] J I Lee J M Skolnik J S Barrett and P C Adamson ldquoAsensitive and selective liquid chromatography-tandem massspectrometry method for the simultaneous quantification ofactinomycin-D and vincristine in children with cancerrdquo Journalof Mass Spectrometry vol 42 no 6 pp 761ndash770 2007

[21] J B Dennison J L Renbarger D O Walterhouse D RJones and S D Hall ldquoQuantification of vincristine and itsmajor metabolite in human plasma by high-performance liquidchromatographytandemmass spectrometryrdquoTherapeutic DrugMonitoring vol 30 no 3 pp 357ndash364 2008

[22] C W N Damen T Israels H N Caron J H M Schellens HRosing and J H Beijnen ldquoValidated assay for the simultaneous

quantification of total vincristine and actinomycin-D concen-trations in human EDTA plasma and of vincristine concentra-tions in human plasma ultrafiltrate by high-performance liquidchromatography coupled with tandem mass spectrometryrdquoRapid Communications in Mass Spectrometry vol 23 no 6 pp763ndash774 2009

[23] G Ling P Zhang J Sun et al ldquoAn LC-MSMS method forsimultaneous determination of vincristine and verapamil in ratplasma after oral administration of a dual agent formulationrdquoBiomedical Chromatography vol 25 no 9 pp 963ndash969 2011

[24] C W N Damen H Rosing J H M Schellens and J HBeijnen ldquoApplication of dried blood spots combined with high-performance liquid chromatography coupled with electrosprayionisation tandem mass spectrometry for simultaneous quan-tification of vincristine and actinomycin-Drdquo Analytical andBioanalytical Chemistry vol 394 no 4 pp 1171ndash1182 2009

[25] E Groninger P Koopmans W Kamps S De Graaf and DUges ldquoAn automated HPLC method to determine intracellularvincristine concentrations inmononuclear cells of childrenwithacute lymphoblastic leukemiardquo Therapeutic Drug Monitoringvol 25 no 4 pp 441ndash446 2003

[26] W Jin and L Jiang ldquoStudy of uptake kinetics of vincristinefor human erythrocytes by capillary zone electrophoresis withelectrochemical detectionrdquoAnalytica ChimicaActa vol 461 no1 pp 117ndash121 2002

[27] A A Nomeir P Kumari M J Hilbert et al ldquoPharmacokineticsof SCH 56592 a new azole broad-spectrum antifungal agent inmice rats rabbits dogs and cynomolgus monkeysrdquoAntimicro-bial Agents and Chemotherapy vol 44 no 3 pp 727ndash731 2000

[28] X J Zhou M Martin M Placidi J P Cano and R RahmanildquoIn vivo and in vitro pharmacokinetics and metabolism ofvincaalkaloids in rat II Vinblastine and Vincristinerdquo EuropeanJournal of DrugMetabolism and Pharmacokinetics vol 15 no 4pp 323ndash332 1990

[29] D A Hamdy and T S Belal ldquoA comparative study of newlydeveloped HPLC-DAD and UHPLC-UV assays for the deter-mination of posaconazole in bulk powder and suspensiondosage formrdquo Journal of Analytical Methods in Chemistry vol2014 Article ID 241035 7 pages 2014



[32] J C Pattersen ldquoA 2-year comparison study of CrlCD BRand HsdSprague-Dawley SD ratsrdquo Fundamental and AppliedToxicology vol 33 pp 196ndash211 1996

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hand estimation of VCR in plasma samples has been carriedout using HPLC-UV detection [17 18] UPLC-MS-MS [19]and several HPLC-MS-MS methods [20ndash23] Besides anHPLC-MS-MS method was presented for the simultaneousdetermination of VCR and actinomycin-D in human driedblood spots [24] In addition quantification of intracellularVCR concentrations in children with acute lymphoblasticleukemia has been described using HPLC with electro-chemical detection procedure [25] Moreover capillary zoneelectrophoresis coupled with electrochemical detection hasbeen employed to study the uptake kinetics of VCR by humanerythrocytes [26] Finally pharmacokinetics of PSZ and VCRin rats have been separately discussed in only two publishedreports [27 28]

Although PSZ-VCR drug-drug interaction has beenexposed in several previous reports the concurrent deter-mination of PSZ and VCR has not been tackled yet sinceno analytical reports can be found in the literature In thisreport we describe the first simple and reliable RP-HPLCwith diode array detection procedure for the simultaneousdetermination of PSZ andVCR in rat plasmaThe structurallyrelated azole antifungal drug (itraconazole ITZ) has beenused as an internal standard The proposed method has beenapplied in a preliminary pharmacokinetics drug interactionstudy in rats An analytical method capable of simultaneouslymeasuring two drugs would be a valuable tool facilitatingthe measurement of pharmacokinetics using a single blooddraw for each time point with no need to split the samplefor processing through two assay methods In serial bloodcollection studies involving rats in particular this is animportant consideration due to the need to keep cumulativeblood volume withdrawal to a minimum

2 Experimental

21 Materials and Reagents Posaconazole and vincristinesulfate powders (purity gt99 for both) were purchasedfrom Selleckchem (Houston TX USA) Itraconazole wasa kind gift from Nifty Labs Pvt Ltd Hyderabad IndiaHPLC grade methanol and acetonitrile (Fisher ScientificUK Limited Loughborough Leicestershire UK) analyti-cal grade potassium dihydrogen orthophosphate (Riedel-de-Haen Germany) and high purity distilled water wereused Noxafil oral suspension labeled to contain 40mgmLPSZ (Patheon Inc Ontario Canada) was purchased fromSchering-Plough SA Vincarine vials labeled to contain1mgmL vincristine sulfate solution (EIMC United Pharma-ceuticals Badr city Cairo Egypt) were obtained from thelocal market

22 Chromatographic Conditions The HPLC-DAD systemconsisted of Agilent 1200 series (autoinjector quaternarypump vacuum degasser and diode array and multiplewavelength detectors G1315 CD and G1365 CD) connectedto a computer loaded with Agilent ChemStation Software(Agilent Technologies Santa Clara CA USA) The DADwavelength was set at 220 and 262 nmThe chromatographicseparationwas achieved using aHC-C18 (46times 250mm 5120583m

particle size) column attached to HC-C18 (46 times 125mm5 120583m particle size) guard column (Agilent TechnologiesSanta Clara CA USA) The gradient elution composed themobile phase acetonitrile and 0015Mpotassiumdihydrogenorthophosphate (30 70 to 80 20 linear over 7 minutes)pumped at 15mLmin Injection volume was 80120583L Alldeterminations were performed at 25∘C

23 Stock and Standard Solutions Stock solutions of PSZand VCR sulphate (100mgL) were separately prepared inmethanol A 10mgL stock solution of the internal standard(ITZ) was prepared in methanol To prepare samples for thecalibration curves and validation assessment three workingstandard solutions (10 1 and 01mgL) of PSZ and VCRwere prepared freshly by successive 110 dilutions of the stocksolutions with methanol

24 Extraction Procedure The ITZ (003mL) was added toeach 02mL rat plasma sample in a glass test tube To therat plasma sample 003mL of 01M NaOH and 6mL ofdiethyl ether were added Because methanol was present inthe standard curve samples an equivalent amount was addedto the test samples as well (02mL) The tubes were coveredvortex-mixed for 2min at high speed and then subsequentlycentrifuged for 10min at sim2500timesg The organic layer wastransferred to new glass tubes and evaporated to drynessin vacuo (Christ rotational vacuum concentrator Germany)The tubes were placed in a minus20∘C freezer until analysis timeThe residues were reconstituted in 100 120583L methanol of which80 120583L volumes were injected into the HPLC

25 Recovery The plasma recoveries were determined forPSZ VCR and ITZ at concentration level 2500 ngmL inrat plasma using four replicates for each concentration Theextraction efficiency was determined by comparing the peakareas of each analyte to the peak areas of the same amountsdirectly injected to the instrument without extraction

26 Validation Calibration curves were constructed usingsamples of 02mL rat plasma containing PSZ VCR and ISThe curve ranged from 50 to 5000 ngmL for both PSZ andVCR The ratios of PSZ and VCR peak areas to IS peak areawere calculated and plotted versus the expected PSZ or VCRconcentrations Owing to the wide range of concentrationsthe calibration curve data were weighed by a factor of 11199092for both drugs Intraday accuracy and precision of the assaywere determined using four sample replicates of 50 500and 2500 ngmL for each of PSZ and VCR in rat plasmain the same day The same concentrations were analyzed inthree separate days for assessment of the interday accuracyand precision For each daily run concentrations weredetermined by comparison with a calibration curve preparedsimultaneously on the same day of analysis Precision wasdetermined using percentage coefficient of variation (CV)and bias was assessed using percentage error of the mean

27 Application to a Drug Interaction PK Study To evaluatethe applicability of this method in vivo two rats (200ndash250 g)






3 Results





VCRITZ

2 4 6 8 10 12 140(min)

0

10

20

30

40

60

50

(mAU

)

PSZ VCR

(mAU

)

04080

120

2 3 4 61 5

(min)

(a)

2 4 6 8 10 12 140(min)

minus100

102030405060

(mAU

)

(b)

minus50minus25

0255075

100125150175190

(mAU

)

2 4 6 8 10 120(min)

(c)










InterdayCV

Interdaymean error




PSZ VCR

R2 = 09982

y = 00007x + 01884

005

115

225

335

4

Peak

area

ratio


R2 = 09966

y = 00006x minus 00581

0

05

1

15

2

25

3

Peak

area

ratio



1

10

100

1000

10000

Con

cent

ratio

n (n

gm

L)

0 20 40 60Time (h)

VCRPSZ

VCRPSZ

1

10

100

1000

10000

0 20 40 60

Con

cent

ratio

n (n

gm

L)

Time (h)



4 Discussion









5 Conclusion





Acknowledgment


References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






3 Results





VCRITZ

2 4 6 8 10 12 140(min)

0

10

20

30

40

60

50

(mAU

)

PSZ VCR

(mAU

)

04080

120

2 3 4 61 5

(min)

(a)

2 4 6 8 10 12 140(min)

minus100

102030405060

(mAU

)

(b)

minus50minus25

0255075

100125150175190

(mAU

)

2 4 6 8 10 120(min)

(c)










InterdayCV

Interdaymean error




PSZ VCR

R2 = 09982

y = 00007x + 01884

005

115

225

335

4

Peak

area

ratio


R2 = 09966

y = 00006x minus 00581

0

05

1

15

2

25

3

Peak

area

ratio



1

10

100

1000

10000

Con

cent

ratio

n (n

gm

L)

0 20 40 60Time (h)

VCRPSZ

VCRPSZ

1

10

100

1000

10000

0 20 40 60

Con

cent

ratio

n (n

gm

L)

Time (h)



4 Discussion









5 Conclusion





Acknowledgment


References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





InterdayCV

Interdaymean error




PSZ VCR

R2 = 09982

y = 00007x + 01884

005

115

225

335

4

Peak

area

ratio


R2 = 09966

y = 00006x minus 00581

0

05

1

15

2

25

3

Peak

area

ratio



1

10

100

1000

10000

Con

cent

ratio

n (n

gm

L)

0 20 40 60Time (h)

VCRPSZ

VCRPSZ

1

10

100

1000

10000

0 20 40 60

Con

cent

ratio

n (n

gm

L)

Time (h)



4 Discussion









5 Conclusion





Acknowledgment


References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






5 Conclusion





Acknowledgment


References












































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

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