Introduction

Platinum complexes are now a well-established class of anti-tumor agents and play an important role in cancer chemotherapy. Pharmacokinetics/pharma-codynamics have been well characterized during the last decade. In oncological practice, doses of anti-cancer agents often adjusted to body surface area, but for many drugs this strategy fails to control drug concentration in individual patients. Since cancer chemotherapy is often associated with high toxic risks, adaptive and therapeutic drug monitoring based on pharmacokinetic parameters may reduce toxicity and even enhance efficacy (Figure 1) Cis-diammine-di-platinum (CDDP) is one of the most important drugs available for the treatment of cancer

in man (1–4). Pharmacokinetic study of cisplatin is complicated by the fact that, following infusion, both protein bound and ultra filterable species of platinum are present in plasma (5). In an aqueous medium the two labile chloride groups of cisplatin can dissociate and aquated platinum (II) products can be formed (6). The aquated species are believed to be reactive forms of cisplatin that produce cova-lent attachment to DNA, the presumed cellular tar-get (7). The protein bound cisplatin has little if any activity (8), although the inactive protein bound platinum can be removed by centrifugal ultrafiltra-tion. There was no significant difference between the ethanol and ultra filtration methods in the unbound platinum concentration (9). Unchanged cisplatin is postulated to be a biologically active species that

Clinical Research and Regulatory Affairs, 2010; 27(1): 1–6

R E S E A R C H A R T I C L E

A simple and sensitive validated HPLC method for quantitative determination of cisplatin in human plasma

K. Harish Kaushik1, Vijay K. Sripuram1, Satish Bedada1, Narsimha Y. Reddy1, G. Indira Priyadarshini2, and Krishna R. Devarakonda3

1Drug Metabolism & Pharmacokinetics Lab, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, India, 2University College of Pharmaceutical Sciences, Andhra University, Vishakapatnam, India, and 3Clinical Research Division, Covidien Inc., Hazelwood, Missouri, USA

AbstractCisplatin is an anti-tumor agent widely employed in cancer chemotherapy. A specific and selective method for the quantitative determination of cisplatin in human plasma and its applications to pharmacokinetic investigations is described. One simple ethanol-induced protein precipitation step followed by simple liq-uid–liquid extraction with chloroform is the only requirement as sample treatment. The resulting solution is injected into a Wakosil II (5 μm, 250 cm × 4.6 mm I.D.) analytical column. The mobile phase consisted of methanol:water:acetonitrile (40:30:30 v/v/v). The limit of quantitation was 1 μg/mL. The method showed good recovery (93.95%) and within batch recovery was 91.59–97.00%. At all levels intra- and inter-assay precision was lower than 7 and 10%, respectively. The intra- and inter-assay accuracy ranged from −2.7 to 2% and from −3.1 to 4.0%, respectively. The selectivity (discrimination between the parent drug and plati-num containing species such as cisplatin metabolites), simplicity and speed of this assay for free cisplatin quantitation should facilitate pharmacokinetic investigations and therapeutic drug monitoring.

Keywords: Cisplatin; HPLC; ethanol; human plasma

Address for Correspondence: Krishna R. Devarakonda, M. Pharm, PhD, FCP, Clinical Research Division, Covidien Inc., 675 McDonnell BlV, Hazelwood, MO 63042, USA. E-mail: krishna.devarakonda@covidien.com

(Received 11 July 2009; revised 04 November 2009; accepted 15 November 2009)

ISSN 1060-1333 print/ISSN 1532-2521 online © 2010 Informa UK LtdDOI: 10.3109/10601330903490462 http://www.informahealthcare.com/crr

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can be determined by the high performance liquid chromatography (HPLC) method. However, ultrafil-terable platinum (the mixture of low molecular mass species) has been generally measured by the atomic absorption spectrometry in the most previous stud-ies. The different disposition behaviors of unchanged cisplatin and ultrafilterable platinum already have been reported. Unchanged cisplatin is eliminated more quickly from plasma than ultrafilterable plati-num, and easily transported to the kidney, which could be linked to nephrotoxicity. Therefore, assay of unchanged cisplatin is significant for managing adverse effects, including nephrotoxicity, in cisplatin chemotherapy.

For both pharmacokinetic investigations and in vitro experiments, it is desirable to have a selec-tive method for the determination of free cisplatin (the active species) because treatment efficacy and toxicity are both related to the concentration of free cisplatin and not to the concentration of other platinum- containing species which may arise from the degradation or metabolism of cisplatin and could interfere in the quantitation of free cisplatin and the evaluation of the efficacy/toxicity. Normal phase and reversed-phase high-performance liquid chromatography (HPLC) systems have been used with a number of detection techniques: UV detec-tion (10, 11), post-column derivatization plus UV detection (12, 13), quenched phosphorescence (14) and electrochemical detection compared to all these methods our method is simple and economical. To fully understand the therapeutic benefits and toxici-ties of various cisplatin dosing regimens, one would ideally like to study the pharmacokinetics of ‘active Pt (II)’ species present in plasma. We report here our development of a reversed-phase HPLC method that has improved selectivity for total ‘active Pt (II)’ species in plasma using ethanol (9) for inducing pro-tein precipitation which is simpler compared to the ultra filtration method.

Materials and methods

Chemicals and reagents

Cisplatin injection vial (Platifirst) was obtained from Cipla Pharma Ltd (Ahmedabad, India), and Sodium diethyl dithio carbamate (DDTC) was supplied by Sigma-Aldrich (St.Louis, USA). Nickel chloride, HPLC grade methanol, acetonitrile, ethanol, and chloro-form were supplied by E.MERCK (Mumbai, India). Double distilled water was used during the entire HPLC procedure. DNS (0.9% w/v) was purchased from Cipla Pharma Ltd.

Instruments and procedures

The HPLC system consisted of LC-10AT VP solvent delivery module (Shimadzu, Kyoto, Japan) and SPD-10AVP UV-Visible spectrophotometric detector, Shimadzu SCL 8A system controller, and Rheodyne injection port (Rheodyne, Cotati, CA) with a 20 µL sample loop. A reverse phase C

18 column (adsor-

bosphere Wakosil II, SGE, Japan Inc., 250 × 4.6 mm, 5 µm) was used, along with a Hamilton Syringe 20 µL (Nevada), Cyclomixer (Remi Equipments, Mumbai, India), Biofuge Fresco centrifuge (Heraeus, Germany), Cooling Centrifuge (Remi Instruments, Mumbai, India), and Accupipet (10–100 µL and 100–1000 µL, Hi Media, Mumbai, India). Mobile phase consisting of methanol:water:acetonitrile (40:30:30 v/v/v) was prepared by addition of specified quantities and mixed thoroughly. Mobile phase was degassed and used for the HPLC analysis; 1.1 mL/min flow rate was main-tained throughout the analysis. The eluent was moni-tored using a UV/VIS detector set at 254 nm. Sensitivity was set at 0.001 absorbance units full scale.

Preparation of standardsTwo independent stock solutions were prepared. One was used for the preparation of the calibration samples and the second for the preparation of the validation samples. The stock solutions of cisplatin were prepared by diluting with saline in a 25.0 mL (polypropylene) volumetric flask to obtain a con-centration of 1000 µg/mL. Calibration samples were prepared by adding the required amount of the stock solutions to human plasma to obtain concentrations of 1, 2, 4, 6, 8, and 10 µg/mL. Validation samples were prepared at low (1.5 µg/mL), medium (5 µg/mL), and high (9 µg/mL) concentration levels. The lower limit of quantitation (LLOQ) was prepared at a concentra-tion of 1 µg/mL. The stock solution of nickel chloride was prepared by dissolving nickel chloride in milli Q water in a 25.0 mL (polypropylene) volumetric flask to obtain a concentration of 1 mg/mL. Validation samples were kept frozen at −20°C until analysis.

Extraction procedure

To each 500 µL of plasma sample, internal stand-ard 10 µL of 300 µg/ml Nickel chloride solution as I.S.(Internal Standard) was added and extracted (18–21) with 1 mL of −20°C ice cold ethanol, vortexed for 1 min, and centrifuged at 13,000 rpm for 15 min and supernatant was collected, to it 50 µL of (10%) DDTC in 0.1 N NaOH was added and incubated in a water bath at 37°C for 25 min and extracted with 200 µL chloroform by vortexing vigorously at maximal speed

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HPLC analysis of cisplatin in human plasma 3

for 1 min, the mixture was separated into aqueous (top), gelatinous (middle), and chloroform ( bottom) layers by centrifugation. Then the chloroform layer was collected and evaporated under vacuum for 10 min and the residue was reconstituted with 50 µL of acetonitrile and 20 µL of the reconstituted sample was injected into the HPLC.

Patient samples and blanksPatient plasma samples were obtained from a patient who participated in a multi-center population phar-macokinetic study of cisplatin (15). The protocol was approved by the Medical Ethics Review Committee of the University Hospital, Warangal, and the patient gave written informed consent. Chemotherapy on the day of treatment consisted of cisplatin (50–100 mg/m2) as a 30 min infusion immediately followed by post-infusion hydration including 1000 mL of Solita T3 for a period of 1 h after the CDDP infusion, or 500 mL of Solita T3 and 500 mL of mannitol for a period of 1 h after the CDDP infusion. Collection of blood samples was started immediately after the cisplatin dose and continued until 19 h. The samples were placed on ice immediately after collection. Subsequently, plasma samples were kept frozen at −20°C until analysis. Blank plasma samples for method validation were obtained from male and female volunteers.

ValidationThe method was validated on linearity, accuracy, recov-ery, freeze-thaw stability, and sample compartment stability. On day 1 the linearity of the calibration curves and the stability in the sample compartment were determined. On the days 2 to 6 precision and accuracy, recovery, and freeze/thaw stability were tested. During validation, six blank samples obtained from six differ-ent human volunteers were tested to demonstrate that there were no interfering components.

LinearityComplete calibration curves were analyzed on 6 sepa-rate days. The three levels of quality control samples were assayed thrice with each standard curve. A linear regression was used to plot the peak area ratio (y) of cisplatin to IS vs cisplatin concentration. The model homoscedasticity was assessed by the Levene test. Best weighting factor for linear regression was determined according to the result of the Levene test and the evo-lution of variance with respect to concentration. Slope, intercept, and correlation coefficient were calculated for each standard curve.

Accuracy and precisionIntra- and inter-day accuracy and precision were evaluated at 1.5, 5, and 9 μg/mL. Six replicates of

each level of quality control samples were assayed in one run for the intra-day experiment. Three replicates of each level of quality control samples were assayed within 6 different days for the inter-day experiment. Accuracy was evaluated by calculating the bias that was determined as ((measured con-centration − theoretical concentration)/(theoretical concentration)) × 100. Precision was evaluated as the coefficient of variation (CV). Acceptance criteria for accuracy and precision were: bias within ±15% and CV lower than 15%. The limit of quantitation (LOQ) was determined as the lowest concentration of cisplatin that could be determined with acceptable accuracy and precision (< 15% for each criterion).

RecoveryRecovery of cisplatin was evaluated at concentration levels corresponding to those of the quality control samples (1.5, 5, and 9 μg/mL). Recovery after protein precipitation was determined by comparing the peak area of the extracted plasma with that of the identi-cal concentration of cisplatin prepared in the mobile phase without extraction. The analysis was carried out on six samples for each level.

Stability during storage in sample compartmentTo determine the freeze–thaw stability of spiked plasma samples, five replicates each of the low, mid-dle, and high QC samples were analyzed after one and two freeze–thaw cycles. The freeze–thaw QC samples were quantified by comparison with a calibration plot prepared after chromatography of freshly spiked samples. For assessment of bench-top stability, the concentrations of low, middle, and high QC samples were determined after 0, 2, and 4 h. The analyte-to-IS peak-area response ratios after 2 and 4 h were com-pared with that after 0 h.

Results and discussion

The extraction of cisplatin was based on protein pre-cipitation technique. Various solvent systems were tried for recovery studies. The maximum recovery was obtained with ice cold ethanol. Hence, ethanol was used for the extraction of drug from plasma, the extraction was carried out in one simple step, which was followed by a chloroform clean-up step, which removed interferences due to plasma and yielded cleaner chromatograms.

Chromatography

Typical chromatograms corresponding to blank plasma and cisplatin containing plasma were given

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in Figures 2 and 3. Chromatogram of patient plasma sample of cisplatin (nickel chloride as I.S.) is shown in Figure 4. No endogenous interference peaks were visible in blank plasma at the retention time of cispla-tin, thereby confirming the specificity of the analyti-cal method. Both the analyte (cisplatin) and the I.S. (nickel chloride) were well separated with retention times of 12.5 and 15.2 min, respectively.

Selectivity

Possible interference from endogenous constituents of human plasma was evaluated by analyzing plasma samples obtained from different donors. Most of the material absorbing at 254 nm eluted before the cisplatin peak. No interfering peaks were observed and no significant peaks were found at the retention

time of cisplatin. Figure 4 shows a representative chromatogram of a human plasma extract after administration of cisplatin. As we can see, potential degradation products of cisplatin were not interfer-ing in the chromatogram and cisplatin was perfectly resolved. This system, therefore, completely sepa-rated cisplatin from endogenous constituents and degradation products.

−1

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Figure 2. Chromatogram of blank plasma (x-axis: Time (min); y-axis: Voltage (mV)).

chromatogram of patients sample(HC21)

-500

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0 5 10 15 20

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Figure 3. Chromatogram of patients plasma sample (HC21) collected from a patient 6 h after administration of 100 mg/m2 cisplatin.

Standard Chromotogram

-500

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V)Figure 4. Typical chromatogram of cisplatin 6 μg/ml in plasma (healthy volunteers).

Cl

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Figure 1. Structure of cisplatin.y = 7.8509x + 0.0979

R2 = 0.9983

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Figure 5. Standard graph of cisplatin in human plasma.

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16TIME vs DV, IPRE

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Figure 6. Concentration time graph generated using NONMEM for population pharmacokinetic study of cisplatin.

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HPLC analysis of cisplatin in human plasma 5

Quantification and linearity

The ratio of peak area of drug (cisplatin) and I.S. was used for the quantification of cisplatin in plasma samples. The calibration curves were linear in the concentration range of 1–10 μg/mL for cisplatin. The calibration/regression equation is y = mx + c, where y represents the peak area ratio of drug to I.S., x represents the concentrations of cispla-tin, m is slope of the curve, and c is the intercept. For linearity assessment, the Levene statistic test showed a significant difference (p < 0.05) between variances of each concentration standard. As the variance grew proportionally to the concentration, the best weighting factor was 1/(peak area ratio). The six standard curves were linear over a concen-tration range of 1 – 10 μg/mL, with a mean slope of 7.85 ± 0.87 (CV = 0.11%) and 0.098 ± 0.34 as intercept (Figure 5). The average coefficient of correlation was 0.997 ± 0.002.

Recovery

Recovery (%) was determined by comparing the analyte-to-IS peak-area ratio for the plasma QC samples (LQC, MQC, and HQC) with the analyte-to-IS peak-area ratio for freshly prepared unextracted aqueous standards containing the same concentra-tions of cisplatin. Total recovery of cisplatin was 93.95% and within-batch recovery was 91.59 – 97.00% (Table 1).

Precision

Intra- and inter-assay precision and accuracy are reported in Table 2. At all levels, intra- and inter-assay precision was lower than 7 and 10%, respectively. The intra- and inter-assay accuracy ranged from −2.7 to 2% and from −3.1 to 4.0%, respectively.

Stability during storage in sample compartment

The freeze–thaw stability ranged from 77.92 – 103.02%. The stability through one and two freeze–thaw cycles was from 85.06 – 103.02% and from 77.92 – 89.76%, respectively. The freeze–thaw stability of cisplatin over two cycles was therefore acceptable (Table 3). To meet acceptance criteria cisplatin must be stable in plasma samples for up to 4 h (16). The bench-top stability ranged between 88.74 – 105.62% (Table 4)

Table 3. Freeze–thaw stability of cisplatin in human plasma.Individual stability (%) LQC MQC HQCOne cycle 1 91.3628 99.823 99.7645 2 78.7324 95.1231 103.2015 3 83.7189 97.3842 110.3246 4 86.4112 89.5673 98.7843 Mean 85.0563 95.4744 103.0187 SD 4.56 3.79 4.5257 CV (%) 5.37 3.97 4.39 n 4 4 4Two cycles 1 85.1245 86.7321 91.8005 2 73.1911 88.2345 93.1295 3 72.8612 82.2265 88.8056 4 80.5162 79.3082 85.3098 Mean 77.9232 84.1253 89.7613 SD 5.1623 3.5528 3.0097 CV (%) 6.62 4.22 3.35 n 4 4 4

Table 1. Bench-top stability of cisplatin in human plasma. % InitialTime (h) LQC MQC HQC0 100 100 1002 99.71 105.62 88.744 101.57 92.75 103.64n 3 3 3

Table 2. Intra- and inter-day precision of determination of cisplatin in human plasma.

Drug

Spiked concentration

(μg/mL)

Mean concentration (μg/mL)

Mean Biasa (%) Precision (%)Cisplatin (inter-day, n = 6)

1.5 1.12 4 9.65 2.13 −1.7 9.99 3.89 −3.1 7.6

Cisplatin (intra-day, n = 6)

1.5 0.98 2 6.25 2.51 −2.7 3.99 4.28 1.9 3.4

a Accuracy is expressed as the bias.

Table 4. Absolute recovery of cisplatin from human plasma. Individual % recovery Total

r ecovery (%)QC LQC MQC HQC1 91.5345 95.7266 97.4632 2 99.6366 88.7573 94.31773 90.3233 95.4163 94.76874 87.4822 90.5341 98.91545 88.9958 95.9528 99.564Mean 91.5945 95.2774 97.0058 93.9592SD 4.7423 2.9701 2.3786 4.0968CV (%) 5.18 3.18 2.45 4.36n 5 5 5 15

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We calculated a lower limit of quantification of 1 µg/mL cisplatin in plasma. As far as we know, the lowest limit of quantitation mentioned in the literature for analysis of cisplatin in saliva by HPLC is 5 µg/mL, although analytical results as low as 11.6 µg/mL car-boplatin in plasma have been reported (5, 11). Despite the fact that cisplatin and carboplatin are chemically not fully identical, we believe that our method is at least 2.5 – 5-times more sensitive than previously reported. Sample size, sample concentration, peak shape, signal-to-noise ratio, and/or injection volume might be relevant parameters, but this was not further investigated.

Conclusions

A simple, specific, and accurate HPLC-UV method has been validated to determine cisplatin concentra-tion in plasma from cancer patients. The method was successfully implemented in routine clinical prac-tice for the population pharmacokinetic analysis of cisplatin. Finally, the simplicity of this assay should facilitate pharmacokinetic research and therapeu-tic drug monitoring of cisplatin in cancer patients. The method has been successfully applied in our laboratory.

Acknowledgement

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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