Determination of the two diastereoisomers of lobaplatin (D-19466) in plasma ultrafiltrate of cancer patients with a normal or an impaired kidney or liver function by high-performance liquid chromatography with ultraviolet detection

Lobaplatin consists of two diastereoisomers, LP-D1 and LP-D2. Being a new cytostatic agent it represents platinum compounds of the third generation and is active in several in vitro tumor models of murine and human origin. To determine the pharmacokinetics of LP-D1 and LP-D2 in cancer patients with and without a normal kidney and liver function, an HPLC procedure was developed and validated. Plasma ultrafiltrate samples were injected into the HPLC system after solid-phase extraction. The standard curves of LP-D1 and LP-D2 in plasma ultrafiltrate were linear over the range 0.071–9.100 and 0.067–8.639 μM, respectively. The recovery from plasma ultrafiltrate was 84% for both diastereoisomers. The within-day accuracy ranged from 98.1 to 100.3% for LP-D1 and from 96.5 to 106% for LP-D2. The between-day accuracy ranged from 99.2 to 101.5% for LP-D1 and 97.7 to 101.2% for LP-D2. The within-day and the between-day precision were ⩽6.0% and ⩽6.1% for LP-D1 and ⩽3.8% and ⩽6.5% for LP-D2, respectively. For pharmacokinetic purposes the method proved to be sufficiently sensitive, specific and accurate for analysing clinical samples.     

Quantification of BNP7787 (dimesna) and its metabolite mesna in human plasma and urine by high-performance liquid chromatography with electrochemical detection

A sensitive and accurate assay was developed and validated to determine BNP7787 (dimesna), a new protector against cisplatin-induced toxicities, and its metabolite mesna in plasma and urine of patients. Both analytes were measured as mesna in deproteinized plasma or in urine diluted with mobile phase using high-performance liquid chromatography with an electrochemical detector provided with a wall-jet gold electrode. The assays for BNP7787 and mesna in deproteinized plasma were linear over the range of 1.6–500 μM and 0.63–320 μM, respectively. In plasma, the mean recovery of BNP7787 over the whole concentration range was 100.6% and of mesna 94.6%. The lower limits of quantitation (LLQs) of BNP7787 and mesna in deproteinized plasma were 1.6 μM and 0.63 μM, respectively. For both compounds the within- and between-day accuracy and precision of the assay was better than 12%. The assays for BNP7787 and mesna in urine were linear over the range of 0.8–1200 μM and 0.63–250 μM, respectively. In urine, the mean recovery of BNP7787 over the whole concentration range was 94.1% and of mesna 93.1%. The LLQ of BNP7787 in urine was 0.8 μM and of mesna 1.6 μM. The within- and between-day accuracy and precision of the assay for BNP7787 and mesna was lower than 15%. The stability of mesna in urine increased with an increasing concentration of mesna, lower temperature and addition of EDTA (1 g/l) and hydrochloric acid (0.2 M). BNP7787 in urine was stable for at least 24 h at temperatures in the range of −20°C up to 37°C and independent of the concentration. The developed assays are currently applied for samples of patients with solid tumors participating in a phase I trial of BNP7787 in combination with cisplatin.     

Determination of dihydroergocryptine in human plasma and urine samples using on-line sample extraction-column-switching reversed-phase liquid chromatography-mass spectrometry

A rapid and sensitive assay for the determination of dihydroergocryptine (DHEC) in human plasma and urine samples with dihydroergotamine (DHET) as the internal standard was developed. The procedure employs on-line sample preparation using an extraction pre-column and an octadecylsilylsilica (ODS) analytical column. After centrifugation human plasma or urine were injected onto the pre-column, concentrated and extracted, back-flushed onto the analytical column and eluted with a binary methanol--aqueous formic acid gradient. Either determination of DHEC as well of its mono- and dihydroxy-metabolites was performed by measurement of the signal responses from MS detection in the selected reaction monitoring (SRM) mode using the transition of the respective parent ions to the common daughter ion at m/z=270.2 amu. The limit of quantitation (LOQ) for determinations of DHEC in both plasma and urine were 25 pg/ml for injected sample volumes of 400 microl. Proportionality of signal responses versus concentration was accomplished within the range of 25-1000 pg/ml. Recovery of target analyte from plasma was 99%. Mean values of the coefficients of variation (CV) for the target analyte in plasma ranged from 1.7 to 13.8% (within-day) and 5.0 to 9.1% (between-day) and accuracy from 91.7 to 102.6% for the within-day and from 95.8 to 98.8% for the between-day measurements. The corresponding values for determinations in urine were 1.7-14.5% (within-day) and 5.3-11.8% (between-day) for CV and 95.8-110.7% (within-day) and 100.1-104.6% (between-day) for accuracy.     

Quantification of mesna and total mesna in kidney tissue by high-performance liquid chromatography with electrochemical detection

A sensitive and selective assay for the determination of mesna and total mesna in tissue was developed and validated. After a simple homogenization, extraction and deproteinization step, mesna could be measured immediately by HPLC with an electrochemical detector provided with a sensitive wall-jet gold electrode. Total mesna (i.e., free mesna and mesna present in mesna disulfides and mixed mesna disulfides) could be measured after pre-column reduction with sodium borohydride to free mesna. The lower limit of quantification of mesna and total mesna was for both compounds 10 nmol/g. The assays for mesna and total mesna in tissue were linear over the ranges of 10-3000 and 10-10000 nmol/g, respectively. The within-day and between-day precisions of both methods were better than 9%. The within-day and between-day accuracy of the mesna assay ranged from 103.7 to 113.6%, whereas the accuracies of the total mesna assay ranged from 97.8 to 106.7%. Mesna in an EDTA containing tissue homogenate or in deproteinized tissue homogenate stored at -80 degrees C was stable for at least 12 weeks. Total mesna was stable under all conditions measured. The developed assays will be applied for the determination of the distribution of mesna and total mesna in tissues of the rat after administration of mesna or BNP7787.     

Determination of cisplatin and cis-diammineaquachloroplatinum(II) ion by liquid chromatography using post-column derivatization with diethyldithiocarbamate

A post-column derivatization method has been developed for the determination of cisplatin and its monohydrated form. Cisplatin was isolated on a strong anion-exchange column, while a strong cation-exchange column was used for the monohydrated complex. Diethyldithiocarbamate was used as reagent and the influence of temperature, pH and methanol content on the yield of derivative was investigated. The reaction was quantitative using a packed-bed reactor with a surrounding temperature of 115°C and a mobile phase consisting of 0.125 M succinic acid—sodium hydroxide buffer pH 5.2 and methanol (2:3, v/v). The resulting complex, Pt(DDTC)₂, was monitored photometrically at 344 nm. The precision of the determination was 11.5% (C.V.) at an injected amount of 20 ng (n = 12) for monoaqua and 8.0% (C.V.) at 9 ng (n = 10) for cisplatin. The method was used to evaluate the plasma concentration of cisplatin and its monohydrated form in a patient.     

New liquid chromatographic assay with electrochemical detection for the measurement of amifostine and WR1065

A high-performance liquid chromatographic method (HPLC) was developed for the analysis of the radio- and chemo-protectant, amifostine and its active metabolite-WR1065 in deproteinized human whole blood and plasma. The two compounds were quantified by measuring WR1065 after two different sample pretreatment procedures. During these procedures, amifostine was quantitatively converted into WR1065, by incubating the sample at 37 degrees C for 4 h at pH<1.0. The resulting amounts of WR1065 were determined by HPLC with coulometric detection (analytical cell: E(1)=200 mV and E(2)=600 mV; guard cell: E(G)=650 mV). The WR1065 standard curve ranged from 0.37 to 50.37 microM. The lower limit of quantitation of WR1065 was 0.25 microM. The within- and between-day precisions were < or = 4.3% and < or = 6.0% for amifostine, < or = 4.4% and < or = 3.8% for WR1065, respectively. The within- and between-day accuracy ranged from 95.4 to 97.7% and 95.4 to 97.8% for amifostine, and from 97.1 to 101.7% and 97.2 to 99.7% for WR1065, respectively. This method minimizes WR1065 loss during sample preparation, and allows for rapid analysis of both compounds on one system. Furthermore, the application of a coulometric electrode is more efficient and requires less maintenance than previously published methods for the two compounds.     

Specific determination of intact cisplatin and monohydrated cisplatin in human plasma and culture medium ultrafiltrates using HPLC on-line with inductively coupled plasma mass spectrometry

We have developed a specific assay for cisplatin in human plasma ultrafiltrate (PUF) and cell culture medium ultrafiltrate (MUF) using HPLC on-line with inductively coupled plasma mass spectrometry (ICP-MS). Separation of cisplatin (6 min) and monohydrated cisplatin (12 min) was achieved using a muBondapak C(18) column (Waters) and a mobile phase (0.075 mM sodium dodecyl sulfate and 3% methanol, adjusted to pH 2.5 with triflic acid) pumped at a flow rate of 0.5 mL/min. The analytes were detected with little background interference by ICP-MS monitoring of platinum masses (m/z 194/195). Calibration curves were linear over three orders of magnitude (0.05-8 microM) and the limit of quantitation was 0.1 microM. Intra- and inter-assay accuracy (range 91.6-113%) and precision (range 1.00-12.3%) were acceptable for PUF and MUF. The method was applied to determining cisplatin during ex vivo incubation of the drug in whole human blood at 37 degrees C. In conclusion, a specific, sensitive and reliable HPLC-ICP-MS assay has been established for determining intact cisplatin in PUF and MUF.     

Development of a liquid chromatography-tandem mass spectrometry assay of six antimicrobials in plasma for pharmacokinetic studies in premature infants

This method provides a simple extraction procedure, as well as a validated, sensitive, and specific liquid chromatography-tandem mass spectrometry assay for the simultaneous quantification of ampicillin, piperacillin, tazobactam, meropenem, acyclovir, and metronidazole in human plasma. The method was validated over concentration ranges specific for each compound, with a lower limit of quantification of 50-300 ng/mL and a sample volume of 50 μL. The method is accurate and precise, with within- and between-day accuracy ranging from 85 to 110% and 92 to 110%, respectively, and within- and between-day precision of 89-111% and 91-109%, respectively. Simplicity, low plasma volume, and high throughput make this method suitable for clinical pharmacokinetic studies in premature infants.     

High-performance liquid chromatographic assay of a new HIV-1 protease inhibitor, LB71350, in the plasma of dogs

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of LB71350 in the plasma of dogs. The analyte was deproteinized with 1.5 volumes of methanol and 0.5 volumes of 10% zinc sulfate, and the supernatant was injected into a 5-μm Capcell Pak C₁₈ column (150×4.6 mm I.D.). The mobile phase was a stepwise gradient mixture of acetonitrile and 0.2% triethylamine–HCl with a flow-rate of 1 ml/min and detection at UV 245 nm. The proportion of acetonitrile was kept at 52% for the first 6 min, increased to 100% for the next 0.5 min, kept at 100% for the next 2 min, decreased to 52% for the next 0.5 min, and finally kept at 52% for the next 7 min. The retention time of LB71350 was 6.9 min. The calibration was linear over the concentration range of 0.1–100 mg/l for dog plasma (r>0.997) and the limit of quantitation was 0.1 mg/l using 0.1 ml plasma. The quality control samples were reproducible with acceptable accuracy and precision at 0.1, 1, 10 and 100 mg/l concentrations. The within-day recovery (n=5) was 90.2–93.9%, the between-day recovery (n=5) was 89.5–93.5%, and the absolute between-day recovery (n=5) was 77–81%. The within-day precision (n=5) and between-day precision (n=5) were 2.59–5.82% and 3.17–4.55%, respectively. No interferences from endogenous substances were observed. Taken together, the above HPLC assay method by deproteinization and UV detection was suitable for the determination of LB71350 in the preclinical pharmacokinetics.     

Determination of Elsamitrucin (BMY-28090) in plasma and urine by high-performance liquid chromatography with fluorescence detection

The cytostatic agent Elsamitrucin is a new fermentation product active in a variety of in vivo tumor models of murine and human origin. To determine its pharmacokinetics during the clinical phase I trial, an HPLC procedure was developed and validated. Plasma samples were extracted after addition of the internal standard, i.e. the analog Chartreusin. Urine samples were injected without extraction of the samples. Because of the wide concentration range of Elsamitrucin in the plasma samples two standard curves were used: up to 100 nM and from 100–1000 nM. Recoveries of Elsamitrucin from plasma were 87% and 74% for concentrations lower and higher than 100 nM, respectively. The detection limits were 1 nM in plasma and 7.5 nM in urine at a signal-to-noise ratio of 3. The accuracy ranged from 95–107% for plasma and from 96–104% for urine. The within-day precision was 4.8% and 2.8% in plasma and urine, respectively. The between-day precision was 4.4% and 7.1% in plasma and urine, respectively. The method proved to be sufficiently sensitive, specific and accurate for analysis of clinical samples for pharmacokinetic purposes.     

High-performance liquid chromatographic assay using electrochemical detection for the combined measurement of amifostine,WR 1065 and the disulfides in plasma

A high-performance liquid chromatographic (HPLC) method was developed for the combined analysis of the chemoprotective agent, amifostine, its active metabolite, WR 1065, and the (symmetrical and mixed) disulfides of WR 1065 in plasma. These three compounds were quantified by measuring WR 1065 after three different sample pretreatment procedures. During these procedures, amifostine and the disulfides were quantitatively converted into WR 1065, by incubating the sample either at a low pH or in the presence of dithiothreitol, respectively. The resulting amounts of WR 1065 were determined by HPLC with electrochemical detection (Au electrode, +1.00 V). The lower limit of quantitation of WR 1065 was 0.15 μM. The within-day and between-day precision were ≤4.4 and ≤8.2% for WR 1065, ≤4.9 and ≤13.1% for amifostine and ≤8.5 and ≤5.5% for the disulfides, respectively. The within-day and between-day accuracy ranged from 97.2 to 109.8% and from 97.6 to 101.5% for WR 1065, from 88.3 to 110.7% and from 99.4 to 101.5% for amifostine and from 99.2 to 110.2% and from 103.3 to 104.9% for the disulfides, respectively. This method is superior to other described methods due to its simple and relatively rapid analysis of all three compounds in one system. Furthermore, it is at least as sensitive as earlier reported methods for one of the compounds and the application of the gold electrode requires only minor maintenance. Therefore, this method is very suitable for pharmacokinetic studies of amifostine and its metabolites. As an example, the plasma concentrations of amifostine, WR 1065 and the disulfides are shown in a patient after receiving an i.v. dose of 740 mg/m² amifostine.     

Quantification of a cytochrome P450 3A4 substrate, buspirone, in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive HPLC-tandem mass spectrometry method was developed for determination of buspirone levels in human plasma. After solid phase extraction and reversed phase HPLC separation, detection of buspirone and the internal standard (prazosin) was performed using eletrospray ionization and selected reaction monitoring in the positive ion mode. Linear calibration curves were established over a concentration range of 0.025-2.5 ng/ml when 0.5 ml aliquots of plasma were used. Satisfactory results of within-day precision (RSD of 1.9-7.7%) and accuracy (% difference of 0.5-6.6%) and between-day precision (RSD of 3.7-11.1%) and accuracy (% difference of 2.2-6.8%) were obtained. The assay has been successfully applied to the analysis of buspirone levels in more than 500 human plasma samples collected from a drug interaction study.     

Quantification of darunavir (TMC114) in human plasma by high-performance liquid chromatography with ultra-violet detection

A precise and accurate high-performance liquid chromatography (HPLC) method with UV detection has been developed and validated for darunavir, a peptidic protease inhibitor. An internal standard, methylclonazepam, was added to 100 microL of plasma before a solid-phase extraction on C18 Bond Elut column. The eluted solutions were evaporated to dryness and reconstituted with 100 microL of mobile phase before being injected in the chromatographic system. The separation was performed on a C8 column using an acetonitrile and ultrapure water mixture (40:60, v/v) as mobile phase. All compounds were detected at a wavelength of 266 nm. The method was linear and validated over a concentration range of 0.25-20mg/L. The within-day precision, ranged from 3.0 to 7.9%, while the within-day accuracy ranged from -11.4 to 0.5%. The between day precision and accuracy were respectively less than 13.7 and -11.4%. The mean recovery was 75.7% for darunavir and 66.7% for methylclonazepam. This method provides a useful tool for therapeutic drug monitoring in HIV patients.     

Diaminonaphtalene, a new highly specific reagent for HPLC-UV measurement of total and free malondialdehyde in human plasma or serum.

We describe a new method to measure free and total malondialdehyde (MDA) in human plasma or serum, which is based on the derivatization of MDA with diaminonaphtalene (DAN) in an acidic medium at 37 degrees C. Derivatization is complete after 180 min at room temperature. By HPLC separation on a C18 column and diode array detection, the diazepinium thus formed exhibits a highly specific UV spectrum with a sharp maximum at 311 nm, which clearly distinguishes MDA from other short-chain aldehydes. Direct injection of deproteinized plasma avoids the use of an internal standard. The between-run imprecision is 9.1% (141 +/- 13 nM) for plasma and 6.6% (658 +/- 44 nM) for a commercial control. Typical within-day imprecision is 8% (93 +/- 7.5 nM) for total MDA, 3.2% (16 +/- 0.5 nM) for free MDA in plasma, and 1.6% (630 +/- 10 nM) for a commercial control. The recovery of MDA added to 10 different plasmas is 93-108% (mean = 100%). Plasma levels in healthy women (n = 79, 45-51 years) are 162 +/- 51 and 24 +/- 15 nM for total and free MDA, respectively. In younger men (n = 19, 21-37 years) total and free MDA are, respectively, 138 +/- 28 and 19 +/- 9 nM.     

A rapid and validated HPLC method to quantify sphingosine 1-phosphate in human plasma using solid-phase extraction followed by derivatization with fluorescence detection

We describe the development and validation of analytical methodology for the determination of sphingosine 1-phosphate (S1P) in plasma. It uses solid-phase extraction (SPE) followed by an automated reversed-phase gradient HPLC column-switching system with a pre-column derivatization with o-phthalaldehyde (OPA) and fluorescence detection. The limit of quantification was determined at 100 ng/ml exogenous sphingosine 1-phosphate with a relative standard deviation for precision and accuracy <15%. The within- and between-day relative standard deviation for precision and accuracy were also less than 15%. This validated method should be suitable to quantify plasma concentration of sphingosine 1-phosphate in relatively large numbers of samples.     

Determination of flavonoids from Orthosiphon stamineus in plasma using a simple HPLC method with ultraviolet detection

A simple liquid chromatographic method was developed for the simultaneous determination of flavonoids from Orthosiphon stamineus Benth, namely sinensitin, eupatorin and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, in plasma. Prior to analysis, the flavonoids and the internal standard (naproxen) were extracted from plasma samples using a 1:1 mixture of ethyl acetate and chloroform. The detection and quantification limits for the three flavonoids were similar being 3 and 5 ng/ml, respectively. The within-day and between-day accuracy values, expressed as percentage of true values, for the three flavonoids were between 95 and 107%, while the corresponding precision, expressed as coefficients of variation, for the three flavonoids were less than 14%. In addition, the mean recovery values of the extraction procedure for all the flavonoids were between 92 and 114%. The calibration curves were linear over a concentration range of 5-4000 ng/ml. The present method was applied to analyse plasma samples obtained from a pilot study using rats in which the mean absolute oral bioavailability values for sinensitin, eupatorin and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone was 9.4, 1.0 and 1.5%, respectively.     

Determination of d-limonene in adipose tissue by gas chromatography-mass spectrometry

We developed a novel method for analyzing d-limonene levels in adipose tissue. Fat samples were subjected to saponification followed by solvent extraction. d-Limonene in the sample extract was analyzed using gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring. Linear calibration curves were established over the mass range of 79.0-2529 ng d-limonene per 0.1g of adipose tissue. Satisfactory within-day precision (R.S.D. 6.7-9.6%) and accuracy (%difference of -2.7 to 3.8%) and between-day precision (R.S.D. 6.0-10.7%) and accuracy (%difference of 1.8-2.6%) were achieved. The assay was successfully applied to human fat biopsy samples from a d-limonene feeding trial.     

Analysis of 12 different pentacyclic triterpenic acids from frankincense in human plasma by high-performance liquid chromatography and photodiode array detection

For the determination of pentacyclic triterpenes of the boswellic acid family in human plasma a novel sensitive method was developed combining serial extraction on diatomaceous earth and graphitized carbon black followed by reversed phase high-performance liquid chromatography (HPLC) and photodiode array detection. The overall average extraction yield of 12 different pentacyclic triterpenic acids was approximately 66%. The calibration graphs were linear with coefficients of correlation for all compounds greater than 0.999. The overall within-day and between-day coefficients of variation (CV) for the 12 pentacyclic triterpenic acids were 5.6 and 6.8%, respectively. This HPLC procedure delivers the analytical sensitivity, precision and accuracy required for clinical pharmacokinetic and therapeutic studies.     

Simple GC-FID method development and validation for determination of alpha-tocopherol (vitamin E) in human plasma

This paper describes the development and validation of a novel GC-FID method for the determination of alpha-tocopherol concentration in human plasma which does not requires derivatization. The standard solutions and the plasma working solutions were prepared in absolute ethanol. To determine the concentration of alpha-tocopherol in human plasma, an aliquot of the plasma sample was deproteinized with ethanol. alpha-tocopherol was extracted with a mixture of hexane and dichloromethane (9:1). GC separation was performed using a HP-5 capillary column. Nitrogen was used as carrier gas at a flow-rate of 2 ml min(-1). Calibration curves were linear over the concentration range 1-30 microg ml(-1) (for standard solutions and solutions without endogenous alpha-tocopherol in plasma) and 5-34 microg ml(-1) (for solutions with endogenous alpha-tocopherol in plasma). Absolute recovery, precision, sensitivity and accuracy assays were carried out. The analytical recovery of alpha-tocopherol from plasma averaged 97.44%. The limit of quantification (LOQ) and the limit of detection (LOD) of method for standard samples were 0.35 microg.ml(-1) and 0.30 microg.ml(-1), respectively. Within-day and between-day precision, expressed as the relative standard deviation (RSD) were less than 4%, and accuracy (relative error) was better than 8%. This novel method, developed and validated in our laboratory, could be successfully applied to the in-vivo determination of alpha-tocopherol. The endogenous alpha-tocopherol amounts in blood of twelve healthy volunteers with no vitamin drug usage were measured with this method.