Simultaneous quantification of pirarubicin, doxorubicin, cyclophosphamide, and vincristine in human plasma of patients with non-Hodgkin's lymphoma by LC-MS/MS method.

Pirarubicin (THP), doxorubicin (DOX), cyclophosphamide (CTX), and vincristine (VCR) are widely used in the treatment of patients with non-Hodgkin's Lymphoma. Herein, a precise and sensitive method was developed for the determination of THP, DOX, CTX and VCR in human plasma by high-performance liquid-chromatography-tandem mass spectrometry (LC-MS/MS). Liquid-liquid extraction was applied to extract THP, DOX, CTX, VCR, and the internal standard (IS, Pioglitazone) in plasma. Agilent Eclipse XDB-C18 (3.0 mm × 100 mm) was utilized and chromatographic separation was obtained in eight minutes. Mobile phases were composed of methanol and buffer (10 mM ammonium formate containing 0.1% formic acid). The method was linear within the concentration range of 1-500 ng/mL for THP, 2-1000 ng/mL for DOX, 2.5-1250 ng/mL for CTX, and 3-1500 ng/mL for VCR. The intra- and inter-day precisions of QC samples were found to be below 9.31 and 13.66%, and accuracy ranged from -0.2 to 9.07%, respectively. THP, DOX, CTX, VCR and the internal standard were stable in several conditions. Finally, this method was successfully utilized to simultaneously determine THP, DOX, CTX and VCR in human plasma of 15 patients with non-Hodgkin's Lymphoma after intravenous administration. Finally, the method was successfully employed in the clinical determination of THP, DOX, CTX, and VCR in patients with non-Hodgkin lymphoma after administration of RCHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone) regimens.

Rapid quantification of vincristine in mouse plasma using ESI-LC-MS/MS: Application to pharmacokinetic studies.

A simple, rapid, and sensitive LC-MS/MS method for determining concentrations of the anticancer alkaloid vincristine in micro volumes of mouse plasma was developed and validated in positive ion mode. Separation of vincristine and the internal standard [2H3]-vincristine was achieved on an Accucore aQ column with a gradient mobile phase delivered at a flow rate of 0.4 mL/min and a run time of 2.2 min. Calibration curves were linear (r2 > 0.99, n = 8) up to 250 ng/mL, with a lower limit of quantitation of 2.5 ng/mL. The matrix effect and extraction recovery for vincristine were ranging 108-110% and 88.4-107%, respectively. The intra-day and inter-day precision of quality controls tested at 3 different concentrations were always less than 15%, and accuracy ranged from 91.7 to 107%. The method was successfully applied to evaluate the pharmacokinetic profile of vincristine in wild-type and CYP3A-deficient mice in support of a project to provide mechanistic insight into drug-drug interactions and to identify sources of inter-individual pharmacokinetic variability associated with vincristine-induced peripheral neuropathy.

A Previously Unknown Drug-Drug Interaction Is Suspected in Delayed Elimination of Plasma Methotrexate in High-Dose Methotrexate Therapy.

Background: High-dose methotrexate (HD-MTX) therapy is widely implemented for leukemia, osteosarcoma, and lymphoma. Although various measures have been taken to avoid toxicity from high serum MTX concentrations, there are many cases of delayed elimination of MTX. Objective: We suspected that delayed elimination of serum MTX was caused by unknown interactions between MTX and concomitant drugs. Methods: Concerning concomitant drugs in the case of delayed elimination of MTX, we performed screening tests in 35 patients who had undergone HD-MTX therapy. We then investigated the risk factors for delayed MTX elimination in 94 patients with leukemia, lymphoma, or osteosarcoma retrospectively. Results: The percentages of concomitant use of Stronger Neo-Minophagen C (SNMC), a glycyrrhizin preparation, and vincristine were higher in the delayed group. The percentage of delayed MTX elimination in patients receiving HD-MTX therapy was 41%. Multiple logistic regression analysis revealed that the concomitant use of SNMC solely was a significant risk factor for delayed MTX (odds ratio = 12.20; 95% CI = 1.06-139.84). Conclusion and Relevance: Concomitant use of SNMC was shown to be related to delayed elimination of serum MTX, and our results suggested a previously unknown drug-drug interaction between MTX and SNMC.

Optimization of rituximab for the treatment of DLBCL: increasing the dose for elderly male patients.

Male sex is associated with unfavourable pharmacokinetics and prognosis in elderly patients with diffuse large B-cell lymphoma (DLBCL). We investigated higher rituximab doses for elderly male DLBCL patients. Elderly patients (61-80 years) received 6 cycles CHOP-14 (cyclophosphamide, doxorubicin, vincristine and prednisone at 14-day intervals) and were randomized to 8 cycles rituximab (males 500 mg/m2 , females 375 mg/m2 ) every 2 weeks or according to an upfront dose-dense schedule. In 268 (120 females, 148 males) no difference between the standard and the upfront dose-dense rituximab schedule was found (3-year PFS 72% vs. 74%; OS 74% vs. 77%; P = 0.651). The 500 mg/m2 dose of rituximab for male patients was associated with serum levels and exposure times slightly better than in females and a male/female hazard ratio of 0.9 for progression-free survival (PFS) and 0.8 for overall survival. For elderly males, 500 mg/m2 was not more toxic than 375 mg/m2 rituximab, but improved PFS by 32.5% (P = 0.039), with a trend for a (30%) better overall survival (P = 0.076) in a planned subgroup analysis adjusting for International Prognostic Index risk factors. We conclude that the higher rituximab dose for elderly male patients abrogated the adverse prognosis of male sex without increasing toxicity. In the era of personalized medicine, sex-specific pharmacokinetics and toxicities should be investigated for all drugs where these parameters impact on outcome.

Prolonged circulation and increased tumor accumulation of liposomal vincristine in a mouse model of rhabdomyosarcoma.

AIM: Our goal was to improve vincristine (VCR) based rhabdomyosarcoma (RMS) therapy by encapsulating the drug into liposomes. A targeting strategy was attempted to enhance tumor accumulation. MATERIALS & METHODS: VCR was loaded in control and peptide-decorated liposomes via an active method. The interaction of an RMS-specific peptide with the presumed target furin and the cellular uptake of both liposomal groups were studied in vitro. Pharmacokinetics and biodistribution of VCR-containing liposomes were assessed in an RMS xenograft mouse model. RESULTS: Liposomes ensured high VCR concentration in plasma and in the tumor. Peptide-decorated liposomes showed modest uptake in RMS cells. CONCLUSION: The investigated peptide-modified liposomal formulation may not be optimal for furin-mediated RMS targeting. Nevertheless, VCR-loaded liposomes could serve as a delivery platform for experimental RMS.

Bioavailability of cyclophosphamide and vincristine after intraperitoneal administration in cats.

Cyclophosphamide and vincristine are widely used intravenous chemotherapeutic agents in both human and veterinary oncology. Although intravenous administration of these chemotherapeutics is the gold standard in most treatment protocols, this route of administration has several disadvantages (e.g. long infusion times and risk of extravasation). Therefore, alternative routes have been explored in the past. Recently, good clinical results were achieved with intraperitoneal (i.p.) administration of cyclophosphamide and vincristine in cats. However, the bioavailability following i.p. administration of cyclophosphamide and vincristine providing proof of principle has not been investigated and is the focus of the present study. The pharmacokinetics of cyclophosphamide and vincristine after i.p. and intravenous administration was investigated in six cats in a cross-over study by analysis of plasma levels of cyclophosphamide and vincristine after simultaneously administration of 0.6 mg/m vincristine and 200 mg/m cyclophosphamide. The median bioavailability on i.p. administration was 76% for cyclophosphamide and 100% for vincristine. Median areas under the curve for i.p. and intravenous administration were 11.4 and 16.0 ng h/ml for cyclophosphamide and 16.7 and 16.5 ng h/ml for vincristine, respectively. No specific i.p. administration-related adverse events were observed after i.p. administration. The high bioavailability of both cyclophosphamide and vincristine after i.p. administration and the absence of specific i.p. administration-related side effects suggest that i.p. administration is a suitable route of systemic chemotherapy for both chemotherapeutics. These results are promising and may serve as a stepping stone for the investigation of the pharmacology, safety, and efficacy of i.p. administration of cyclophosphamide and vincristine in humans.

Clinical pharmacokinetics and effects of vincristine sulfate in dogs with transmissible venereal tumor (TVT).

This study was conducted to evaluate the pharmacokinetic characteristics of vincristine and their correlation with its clinical effects in dogs with transmissible venereal tumor (TVT). Dogs with TVT were intravenously administered vincristine sulfate at a dose of 0.7 mg/m(2) of body surface area. Blood samples were collected starting from 5 min to 48 hr after drug administration. The plasma concentration of vincristine was determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The pharmacokinetic parameters of vincristine were characterized using a two-compartmental pharmacokinetic model. The volume of distribution, distribution half-life, elimination half-life and plasma clearance were 0.660 ± 0.210 l/kg, 21.5 ± 6.90 min, 47.6 ± 14.2 min and 0.010 ± 0.001 l/min/kg, respectively. Tumor regression was determined at weekly interval by a physical examination and histopathological analysis. In our study, three to eight administrations of vincristine at a dose of 0.7 mg/m(2) were able to induce a complete tumor regression without any evidence of gross lesion of disease. Therefore, this investigation provides the pharmacokinetic characteristics of vincristine in dogs with TVT, which may be used as an integration tool to gain a better understanding of the disposition properties of the drug and the correlation of these properties with the drug's clinical effects. In addition, we validated the LC-MS/MS method and found that it is suitable for the pharmacokinetic study of vincristine in dog plasma.

Determination of free and total vincristine in human plasma after intravenous administration of vincristine sulfate liposome injection using ultra-high performance liquid chromatography tandem mass spectrometry.

Vincristine sulfate liposome is a liposomal formulation of vincristine sulfate, a traditional anticancer drug, encapsulated in the aqueous core of phospholipid/cholesterol liposomes, which are kinds of targeted carriers to enhance malignancy targeting, exposure and anticancer activity of the drug. To evaluate and compare the pharmacokinetics of nonliposomal and liposome-encapsulated VCR and pharmacodynamic relationships associated with the toxicity and the efficacy behavior, it is essential to have a reliable method of separating the free and liposomal forms of the drug. In this paper, we have developed and validated methods to quantify the free vincristine (F-VCR) and total vincristine (T-VCR) in human plasma after intravenous administration of vincristine sulfate liposome injection (VSLI). The methods involve solid-phase extraction (SPE) for separating the F-VCR and liquid-liquid extraction (LLE) for releasing the VCR totally from the liposomal forms followed by an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode using positive electrospray ionization (ESI). The methods were validated over the concentration range of 0.2-50 ng/mL for F-VCR and 0.5-400 ng/mL for T-VCR, respectively. Inter- and intra-day precision (RSD%) were ≤4.7% for F-VCR and ≤9.8% for T-VCR, respectively. The accuracies were between -2.3 and 9.1% for F-VCR and between -3.2 and 6.9% for T-VCR, respectively. The extraction recovery and the matrix effect were investigated. The methods were successfully applied to the pharmacokinetic study of VSLI in Chinese subjects with lymphoma.

Pharmacokinetic characteristics of vincristine sulfate liposomes in patients with advanced solid tumors.

AIM: To evaluate the single- and multiple-dose pharmacokinetics of vincristine sulfate liposomes (VSLI) in patients with advanced solid tumors. METHODS: In single-dose pharmacokinetic study, 16 patients were administered VSLI (1.5, 2.0, or 2.3 mg·m(-2)) through intravenous infusion. Another 6 patients receiving vincristine sulfate (VCR, 2.0 mg) were taken as the control. In multiple-dose pharmacokinetic study, 12 patients were administered VSLI (1.5 or 1.8 mg·m(-2)) through intravenous infusion weekly for 4 consecutive weeks. The plasma concentration of VSLI was determined using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. RESULTS: After intravenous infusion of the single dose of VSLI, the plasma concentrations were characterized by bi-exponential decline curves. No statistically significant differences were observed between the main pharmacokinetic parameters in the 3 dose groups. Compared with the patients receiving VCR, the patients treated with VSLI displayed an increase in the area under the plasma concentration vs time curve (AUC), and a decrease in plasma clearance rates. On the 4th cycle in the multiple-dose study, the plasma concentration of VCR in all subjects prior to the weekly administration was below the lower limit of quantification (LLOQ). The calculated pharmacokinetic parameters from the subjects in the multiple- and single-dose (1.5 mg·m(-2)) groups had no significant differences. Although the administration of liposomal VCR may significantly elevate the plasma concentration of VCR, VSLI-associated adverse events were similar to those associated with conventional VCR. CONCLUSION: VSLI exhibits a lower clearance and a higher AUC compared with conventional VCR. No accumulation was observed in patients exposed to VSLI for 4 consecutive weeks. VSLI was generally tolerated in the subjects. The phase II dose of VSLI may be recommended as 4 doses of 1.5 mg·m(-2) for treatment of patients with advanced solid tumors.

Impact of plasma and intracellular exposure and CYP3A4, CYP3A5, and ABCB1 genetic polymorphisms on vincristine-induced neurotoxicity.

PURPOSE: The aim of this study was to investigate the impact of plasma and intracellular exposure and CYP3A4, CYP3A5, and ABCB1 polymorphisms on vincristine neurotoxicity. We subsequently assessed the impact of ABCB1 polymorphisms on intracellular vincristine accumulation. METHODS: Children treated for solid tumors were enrolled in the study (n = 26) and received 1.5 mg/m² of vincristine per course. Individual pharmacokinetic parameters and CYP3A4, CYP3A5, and ABCB1 genotypes were available from a previous analysis. A global toxicity score (pain, peripheral neurotoxicity, and gastrointestinal toxicity) was collected at each course. Vincristine in plasma and PBMCs were quantified by LC-MS/MS. RESULTS: Vincristine plasma and intracellular concentrations ranged from 0.40 to 89.6 ng/ml and from 0.00225 to 1.85 ng/10(6) cells over a 24-h interval, respectively. The global toxicity score ranged from 0 to 6 and was not correlated with individual pharmacokinetics parameters. Neurotoxicity events (global score ≥ 3) were observed in 8 patients but the incidence was not influenced by the different studied polymorphisms. The global toxicity score was correlated with age, body surface area, and dose in mg. A trend to higher intracellular/plasma ratio of vincristine was found for patients with heterozygous diplotype (CGC-TTT) of ABCB1. CONCLUSIONS: None of the different genetic covariates nor plasma and intracellular exposure was predictive of the observed neurotoxicity in our pediatric population. Nevertheless, the heterozygote diplotype of ABCB1 appears to influence the intracellular accumulation of vincristine. Owing to the small sample size, further evaluations are needed in a larger patient cohort.

An LC-MS/MS method for simultaneous determination of vincristine and verapamil in rat plasma after oral administration of a dual agent formulation.

A rapid and sensitive method for simultaneous determination of vincristine and verapamil in rat plasma was first developed and validated, using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode via electrospray ionization (ESI). The method, which required a small sample volume (25 µL) of plasma, was linear in the concentration range of 0.5-500 ng/mL for vincristine and 0.1-100.0 ng/mL for verapamil. Finally, the method was successfully employed in a pharmacokinetic study of vincristine and verapamil in rats after an oral administration of a dual-agent formulation containing vincristine and verapamil.

Pharmacodynamic and pharmacokinetic study of pegylated liposomal doxorubicin combination (CCOP) chemotherapy in patients with peripheral T-cell lymphomas.

AIM: To investigate the pharmacodynamic and pharmacokinetic parameters of pegylated liposomal doxorubicin (PLD) combined with cyclophosphamide, vincristine, and prednisolone in patients with peripheral T-cell lymphomas (PTCL). METHODS: Seven chemonaive patients and four patients with relapsed peripheral T-cell lymphomas were treated with a CCOP regimen consisting of an intravenous administration of cyclophosphamide (750 mg/m(2)), vincristine (1.4 mg/m(2)), and PLD (30 mg/m(2)) on d 1, as well as an oral administration of prednisolone (60 mg/m(2)) on d 1-5. This regimen was repeated every 3 weeks for six cycles, and the clinical response and toxicity of the regimen were monitored. In addition, the plasma concentration of PLD at different time points was determined before and after treatment. The pharmacokinetics (PKs) software was used to estimate the pharmacokinetic parameters of PLD. RESULTS: The 11 PTCL patients received 35 treatment cycles. Three of them achieved complete response (CR), two partial response (PR), four stable disease (SD), and two progressive disease (PD). The overall response rate (ORR) was 45.5%, and the CR rate was 27.3%. In the 7 chemonaive patients, three achieved CR, two PR, one SD, and one PD. The ORR was 71.4%, and CR rate was 42.9%. The median follow-up time was 15 months, but 6 out of 11 patients were dead at the time of data analysis. The 1-year overall survival rate was 45.5%, and the median progression-free survival (PFS) rate was 6.5 [95% confidence interval (95% CI) 3.17-19.02] with a survival rate of 11.5 months (95% CI 6.65-16.36). The main toxicity was myelosuppression. Oral mucositis and hand-foot syndrome seldom occurred. The PLD plasma concentration from nine patients ranged from 1.7036 to 9.2207 mg·L(-1) after administration of the CCOP regimen (0-168 h). The pharmacokinetic parameters AUC(0-∞), CL, t(1/2), and V(d) were 910.76 mg/L·h, 0.043 L·h(-1)·m(-2), 68.40 h, and 3.56 L/m(2), respectively. CONCLUSION: The CCOP regimen was effective and well tolerated in patients with peripheral T-cell lymphomas. The results of the pharmacokinetic parameters showed that PLD had long retention time in blood circulation.

Pharmacokinetics and safety of Marqibo (vincristine sulfate liposomes injection) in cancer patients with impaired liver function.

Marqibo (vincristine sulfate liposome injection, VSLI) is a novel liposomal formulation of vincristine sulfate (VCR) being developed for the systemic treatment of cancer. This study evaluated the pharmacokinetics (PK) of Marqibo in subjects with melanoma and impaired hepatic function. Calculated PK parameters were similar in subjects with impaired liver function compared with those in subjects with adequate liver function. Subjects with impaired liver function universally had a monoexponential total plasma VCR concentration versus time decline, whereas two thirds of subjects with adequate liver function had a biexponential decline profile. Because one third of subjects with normal hepatic function demonstrated monoexponential disposition, lack of biexponential disposition in the hepatically impaired subjects cannot be clearly attributed to liver impairment. VSLI was generally well tolerated in all subjects.

Validation of an electrospray ionization LC/MS/MS method for quantitative analysis of vincristine in human plasma samples.

Vincristine is a natural vinca alkaloid widely used in paediatric cancer treatment. Vincristine pharmacokinetics has been already studied, but few data are available in paediatric populations. A sensitive and specific liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed for the quantification of vincristine in plasma in order to investigate pharmacokinetics in a paediatric population. Two hundred microliters of plasma was added to vinblastine, used as internal standard. Chromatographic separation was achieved on a C8 HPLC column (Phenomenex Luna 50 mm x 2.0 mm, 3.0 microm) with a mobile phase gradient at a flow rate of 0.2 ml/min. Quantification was performed using the transition of 825.4-->765.4 (m/z) for vincristine and 811.4-->751.4 (m/z) for vinblastine. Chromatographic separation was achieved in 8 min. The limit of quantification was 0.25 ng/ml with a precision of 10.2% and an accuracy of 99.6%. The calibration curve was linear up to 50.0 ng/ml. Intra-day precision and accuracy ranged from 6.3% to 10% and from 91.9% to 100.8%, respectively. Inter-assay precision and accuracy ranged from 3.8% to 9.7% and from 93.5% to 100.5%, respectively. No significant matrix effect was observed for vincristine. A rapid, specific and sensitive LC/MS/MS method for quantification of vincristine in human plasma was developed and is now successfully applied for pharmacokinetic studies in paediatric patients.

Diet-induced obesity alters vincristine pharmacokinetics in blood and tissues of mice.

Obesity is associated with poorer outcome from many cancers, including leukemia. One possible contributor to this could be suboptimal chemotherapy dosing in obese patients. We have previously found that vincristine (VCR) is less effective in obese compared to non-obese mice with leukemia, despite weight-based dosing. In the present study, we administered (3)H-VCR to obese and control mice to determine whether obesity would cause suboptimal VCR exposure. Blood VCR concentrations were fitted with a three-compartment model using pharmacokinetic analysis (two-stage PK) in three subsets of VCR concentrations vs. time method. Tissue and blood VCR concentrations were also analyzed using non-compartmental modeling. Blood VCR concentrations showed a triexponential decay and tended to be slightly higher in the obese mice at all time-points. However, the t(1/2,beta) and t(1/2,gamma) were shorter in the obese mice (9.7 min vs. 44.5 min and 60.3h vs. 85.6h, respectively), resulting in a lower AUC(0-infinity) (13,099 ng/m Lh vs. 15,384 ng/mL h). Had the dose of VCR been "capped", as is done in clinical practice, the AUC(0-infinity) would have been 36% lower in the obese mice than the controls. Tissue disposition of VCR revealed a biexponential decay from spleen, liver, and adipose. Interestingly, VCR slowly accumulated in the bone marrow of control mice, but had a slow decay from the marrow in the obese mice. Thus, obesity alters VCR PK, causing a lower overall exposure in circulation and bone marrow. Given the high prevalence of obesity, additional PK studies should be performed in obese subjects to optimize chemotherapy dosing regimens.

Plasma levels of copper, manganese and selenium in an adult population in southern Spain: influence of age, obesity and lifestyle factors.

Copper, manganese and selenium are elements involved in protecting the body against oxidative stress. Determining their plasma level may contribute to assessing the health and nutritional status of populations. The aim of this study was to assess factors influencing copper, manganese and selenium plasma levels in an adult Mediterranean population and to identify groups at risk of deficiency. A cross-sectional survey was carried out in Andalusia, a region in southern Spain. Blood samples were obtained in a random subsample of 340 subjects. Food consumption was assessed by 48-h recall. Height, weight, skinfolds, waist and hip circumferences were measured. Copper, manganese and selenium were measured in plasma. Information about physical exercise, educational level, alcohol and smoking habits was obtained with a structured questionnaire. Plasma copper was found to be higher in women than among men. Hypocupraemia was found in 4.4% of the population, while 9.7% presented hypomanganesemia. Moreover, 86.5% presented plasma selenium values below 125microg/L (cutoff for optimal glutathione peroxidase activity). No association was found between plasma elements, anthropometric indices and lifestyle factors; there were tendencies, no more. Copper tended to decrease in obese and increase in sedentary, while selenium tended to decrease among smokers. Plasma Cu was positively correlated with the consumption of monounsaturated and polyunsaturated fats. Plasma Mn was directly correlated with the consumption of dairy products. Levels of Se were positively correlated with age, the consumption of fruit, vegetables, energy obtained from carbohydrates, and the consumption of fibre, and inversely correlated with the consumption of meat and sweets. Our results provide an estimate of the copper, manganese and selenium status in the adult population of southern Spain. The correlations found for Se suggest that there is a tendency for Se levels to be better maintained among the population that shows a stronger preference for the traditional diet.

Vincristine pharmacokinetics is related to clinical outcome in children with standard risk acute lymphoblastic leukemia.

Vincristine is a key drug in the treatment of childhood and adult acute lymphoblastic leukemia (ALL), and many other childhood malignancies. Despite decades of wide clinical use, no data on the correlation between vincristine pharmacokinetics and long-term clinical outcome have been published. We here report clinical data (median follow-up time 10.5 years, range 7.3-12 years) for 86 children with B-cell precursor ALL, in whom vincristine kinetics were studied on treatment day 1. The median total plasma clearance was 429 and 331 ml/min per m(2) and the area under the plasma concentration-time curve (AUC) was 4.49 and 5.40 mg/l x min in relapse and non-relapse patients, respectively (not significant). In standard risk patients, where treatment depends more heavily on vincristine than in other subgroups, the relative risk (RR) of relapse was significantly increased for patients with clearance values above median (RR 5.2; P = 0.036), or AUC values below median (RR 5.8; P = 0.025). Our data suggest a relationship between the antileukemic effect and the systemic exposure of the drug, which warrants further studies.

Quantification of vincristine and its major metabolite in human plasma by high-performance liquid chromatography/tandem mass spectrometry.

An analytical method using electrospray ionization and high-performance liquid chromatography/tandem mass spectrometry (LC/ESI-MS/MS) was developed to quantify vincristine and M1, the CYP3A-mediated metabolite of vincristine, in human plasma. Vinblastine (internal standard), vincristine, and M1 in plasma were extracted in methylene chloride after acidification with TCAA. The analytes were separated on an Inertsil ODS-3 C18 column (2.1 x 150 mm) with a 5-mum particle size using a gradient elution with a run time of 20 min. The initial mobile phase composition was 0.2% formic acid/water (80:20, v/v) with a final composition of 0.2% formic acid/water (20:80, v/v). Detection was accomplished with multiple reaction monitoring for vinblastine (m/z 406.3--> 271.7), vincristine (m/z 413.2--> 362.2), and M1 (m/z 397.3 --> 376.2). At three concentrations of vincristine and M1, the inter-day and intra-day accuracy and precision were within the acceptable limits for validation (106.8 +/- 9.6% for intra-day, n = 5 each concentration; 90.9 +/- 10.9% for inter-day, n = 4 each concentration). For both vincristine and M1, the concentration limits of quantification and detection were 12 pg/mL and 6 pg/mL, respectively. Stability studies indicated that 80% of M1 degraded in plasma after 15 hours at room temperature (n = 3, high and low QC concentrations). Therefore, short plasma processing times (<30 min) are recommended. The assay was used successfully to quantify vincristine and M1 in pediatric plasma samples up to 24 hours after vincristine administration. Vincristine and M1 concentrations were within the limits of quantification for all patient plasma samples.

Rapid and sensitive analysis of vincristine in human plasma using on-line extraction combined with liquid chromatography/tandem mass spectrometry.

A simple and rapid method has been developed and validated for the quantitation of vincristine in human plasma by liquid chromatography/tandem mass spectrometry (LC/MS/MS) with atmospheric pressure chemical ionization using on-line solid-phase extraction. The method uses vinblastine as internal standard and the sample preparation is limited just to a plasma protein precipitation step. Further sample clean-up is carried out on-line through a perfusion column preceding an analytical phenyl LC column, the latter directly connected to the mass spectrometer. Quantitation is performed in multiple reaction monitoring mode using the transitions of m/z 825.3 --> 765.3 and 811.3 --> 751.3 for vincristine and vinblastine respectively. The assay was linear (r2 > or =0.99) in a concentration range from 0.1 to 500 ng/mL. Carry-over, measured on the experimental set-up, was less than 0.04%. Recovery for vincristine and the internal standard was within 90-95%. The intra-day and inter-day assay precision ranged from 1.2% to 6.8% RSD while mean percentage deviation from nominal value ranged from 0.01% to 6.1%. The proposed assay was found suitable for pharmacokinetics investigations and clinical therapeutic drug monitoring especially in pediatric cancer patients.