Quantitative high-performance liquid chromatographic determination of acrolein in plasma after derivatization with Luminarin 3

A rapid, sensitive and specific high-performance liquid chromatographic method for the quantification of acrolein (1), one of the toxic metabolites of oxazaphosphorine alkylating agents (cyclophosphamide and ifosfamide) was developed. Condensation of acrolein with Luminarin^® 3 afforded a fluorescent derivative that could be specifically detected and quantified. Chromatographic conditions involved a C₁₈ RP column Uptisphere and a gradient elution system to optimize resolution and time analysis. The method showed high sensitivity with a limit of detection of 100 pmol/ml and a limit of quantification of 300 pmol/ml. This technique is particularly suitable for pharmacokinetic studies on plasma of oxazaphosphorine-receiving patients.     

A liquid chromatographic-tandem mass spectrometric method for determination of artesunate and its metabolite dihydroartemisinin in human plasma

A bioanalytical method for the analysis of artesunate and its metabolite dihydroartemisinin in human plasma using high throughput solid-phase extraction in the 96-wellplate format and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. The method was validated according to published FDA guidelines and showed excellent performance. The within-day and between-day precisions expressed as RSD, were lower than 7% at all tested concentrations including the lower limit of quantification. Using 50 microl plasma the calibration range was 1.19-728 ng/ml with a limit of detection at 0.5 ng/ml for artesunate and 1.96-2500 ng/ml with a limit of detection at 0.6 ng/ml for dihydroartemisinin. Using 250 microl of plasma sample the lower limit of quantification was decreased to 0.119 ng/ml for artesunate and 0.196 ng/ml dihydroartemisinin. Validation of over-curve samples in plasma ensured that accurate estimation would be possible with dilution if samples went outside the calibration range. The method was free from matrix effects as demonstrated both graphically and quantitatively.     

Direct gas chromatographic determination of dechloroethylcyclophosphamide following microsomal incubation of cyclophosphamide

A method for the sensitive determination of dechloroethylcyclophosphamide (3-DCl) in microsomal incubation mixtures was developed. 3-DCl, a side-chain oxidation product of cyclophosphamide (CP), was isolated by extraction with acetic acid ethyl ester following solid-phase extraction on C₈ cartridges. Quantification of the metabolite was performed by direct capillary gas chromatography with a nitrogen-phosphorus detector without prior derivatization. The method showed good sensitivity and reproducibility with a detection limit of 1 ng/ml and a limit of quantification of 5 ng/ml. The suitability of the method is shown for the quantification of 3-DCl following incubation of CP with human liver microsomes.     

Determination of tezosentan,a parenteral endothelin receptor antagonist,in human plasma by liquid chromatography-tandem mass spectrometry

An analytical method was developed for the quantification of tezosentan in human plasma obtained in clinical studies. The method was linear in the range 1 to 512 ng/ml. After liquid-liquid extraction, the samples were analyzed by reversed-phase HPLC with tandem mass spectrometry. The limit of quantification was 1 ng/ml and the extraction recovery was at least 88.2%. Intra- and inter-assay coefficients of variation were below 10%. Stability tests revealed that tezosentan is stable under the different conditions tested.     

Development and validation of a high-performance liquid chromatography method using diode array detection for the simultaneous quantification of aripiprazole and dehydro-aripiprazole in human plasma

A high-performance liquid chromatography method with diode array detection (HPLC-DAD) was developed for quantification of aripiprazole and dehydro-aripiprazole, in human plasma. After a simple liquid-liquid extraction, chromatographic separation was carried out on a C18 reversed-phase column, using an ammonium buffer-acetonitrile mobile phase (40:60, v/v). The total run time was only 7 min at a flow-rate of 1.0 ml/min. The precision values were less than 12% and the accuracy values were ranging from 98 to 113% and the lower limit of quantification was 2 ng/ml for both compounds. Calibration curves were linear over a range of 2-1000 ng/ml. The mean trough plasma concentrations in patients treated with aripiprazole were 157 and 29 ng/ml for aripiprazole and dehydro-aripiprazole, respectively.     

Simple high-performance liquid chromatographic method for determination of ketoconazole in human plasma

A simple high-performance liquid chromatographic method using fluorescence detection was developed for the determination of ketoconazole in human plasma. The method entailed direct injection of the plasma sample after deproteinization using acetonitrile. The mobile phase comprised 0.05 M disodium hydrogen orthophosphate and acetonitrile (50:50, v/v) adjusted to pH 6. Analysis was run at a flow-rate of 1.5 ml/min with the detector operating at an excitation wavelength of 260 nm and an emission wavelength of 375 nm. The method is specific and sensitive with a quantification limit of approximately 60 ng/ml and a detection limit of 40 ng/ml at a signal-to-noise ratio of 3:1. Mean absolute recovery value was about 105%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 14%. The calibration curve was linear over a concentration range of 62.5–8000 ng/ml.     

Simple high-performance liquid chromatographic method for the determination of metformin in human plasma

A simple high-performance liquid chromatographic method using ultraviolet detection was developed for the determination of metformin in human plasma. The method entailed direct injection of the plasma sample after deproteination using perchloric acid. The mobile phase comprised 0.01 M potassium dihydrogen orthophosphate (pH 3.5) and acetonitrile (60:40, v/v). Analyses were run at a flow-rate of 1.0 ml/min with the detector operating at a detection wavelength of 234 nm. The method is specific and sensitive, with a quantification limit of approximately 60 ng/ml and a detection limit of 15 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery value was about 97%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 8%. The calibration curve was linear over a concentration range of 62.5–4000 ng/ml.     

Determination of Oltipraz in serum by high-performance liquid chromatography with optical absorbance and mass spectrometric detection

Three methods have been developed for the analysis of Oltipraz in serum. A method suitable for routine use employs spiking with a homologous internal standard, off-line solid-phase extraction, high-performance liquid chromatographic separation, and optical absorbance detection at 450 nm. Method detection limit is about 1 ng/ml. A second method, less susceptible to bias from co-eluting interferences, uses a stable isotope-labeled internal standard, similar extraction and separation, and detection by thermospray mass spectrometry. Method detection limit is about 0.2 ng/ml. A third method was developed which can be used without specially synthesized internal standards. It uses on-line solid-phase extraction, with quantification by comparison with external standards. Method detection limit is about 3 ng/ml. Good agreement was observed between these methods and with similar and different methods run in other laboratories. Calibration curves were linear over the entire range which was investigated, i.e., up to 500 ng/ml. Coefficients of variation were similar for all three methods, being about 5%.     

Development of a sensitive liquid chromatography method coupled with a tandem mass spectrometric detection for the clinical analysis of vinflunine and 4-O-deacetyl vinflunine in blood, urine and faeces

A sensitive and specific liquid chromatographic method coupled with tandem mass spectrometric detection was set up and fully validated for the simultaneous quantification of vinflunine (VFL) and its pharmacologically active metabolite, 4-O-deacetyl vinflunine (DVFL). The two compounds, as well as vinblastine (used as internal standard), were deproteinised from blood and faeces, analysed on a cyano type column and detected on a Micromass Quattro II system in the positive ion mode after ionisation using an electrospray ion source. In blood, linearity was assessed up to 200 ng/ml for vinflunine and 100 ng/ml for 4-O-deacetyl vinflunine. The lower limit of quantification was validated at 250 pg/ml for both compounds. In other biological media, the linearity was assessed within the same range; the limit of quantification was adjusted according to the expected concentration levels of each compound. This method was first developed in order to identify the structures and to elucidate the metabolic pathway of vinflunine. Thanks to its high sensitivity and specificity, the method has enabled the quantification of vinflunine and 4-O-deacetyl vinflunine in blood at trace levels, and has contributed to the knowledge of vinflunine metabolism by monitoring up to 10 metabolites.     

Determination of amiodarone and desethylamiodarone in horse plasma and urine by high-performance liquid chromatography combined with UV detection and electrospray ionization mass spectrometry

A rapid method for the quantification of amiodarone and desethylamiodarone in animal plasma using high-performance liquid chromatography combined with UV detection (HPLC-UV) is presented. The sample preparation includes a simple deproteinisation step with acetonitrile. In addition, a sensitive method for the quantification of amiodarone and desethylamiodarone in horse plasma and urine using high-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is described. The sample preparation includes a solid-phase extraction (SPE) with a SCX column. Tamoxifen is used as an internal standard for both chromatographic methods. Chromatographic separation is achieved on an ODS Hypersil column using isocratic elution with 0.01% diethylamine and acetonitrile as mobile phase for the HPLC-UV method and with 0.1% formic acid and acetonitrile as mobile phase for the LC-MS/MS method. For the HPLC-UV method, good linearity was observed in the range 0-5 microg ml(-1), and in the range 0-1 microg ml(-1) for the LC-MS/MS method. The limit of quantification (LOQ) was set at 50 and 5 ng ml(-1) for the HPLC-UV method and the LC-MS/MS method, respectively. For the UV method, the limit of detection (LOD) was 15 and 10 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs of the LC-MS/MS method in plasma were much lower, i.e. 0.10 and 0.04 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs obtained for the urine samples were 0.16 and 0.09 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The methods were shown to be of use in horses. The rapid HPLC-UV method was used for therapeutic drug monitoring after amiodarone treatment, while the LC-MS/MS method showed its applicability for single dose pharmacokinetic studies.     

Simple, sensitive and rapid LC-MS method for the quantitation of indapamide in human plasma--application to pharmacokinetic studies

A sensitive and specific method using liquid chromatography with electrospray ionization mass spectrometry (LC-ESI-MS) has been developed and validated for the identification and quantification of indapamide in human plasma. A simple liquid-liquid extraction procedure was followed by injection of the extracts on to a C18 column with gradient elution and detection using a single quadrupole mass spectrometer in selected ion monitoring (SIM) mode. The method was tested using six different plasma batches. Linearity was established for the concentration range 0.5-100.0 ng/ml, with a coefficient of determination (r) of 0.9998 and good back-calculated accuracy and precision. The intra- and inter-day precision (RSD%) was lower than 10%, and accuracy ranged from 85 to 115%. The lower limit of quantification was reproducible at 0.2 ng/ml with 0.2 ml plasma. The proposed method enables the unambiguous identification and quantification of indapamide for pre-clinical and clinical studies.     

Quantification of steroid glycosides from Hoodia gordonii in porcine plasma using high performance liquid chromatography-mass spectrometry

An HPLC-ESI-MS/MS method using collision induced dissociation - multiple reaction monitoring was developed for the quantification of eight Hoodia gordonii steroid glycosides and their metabolites in porcine plasma samples. The method was validated for the three most important glycosides and was successfully applied also for the related glycosides and metabolites. The limits of quantification were 0.04 ng ml(-1) for the two main steroid glycosides and 0.1 ng ml(-1) for the detiglated metabolites. These limits are sufficiently low to allow monitoring the concentration-time profiles in plasma after feeding H. gordonii. The standard deviations of the intra-day measurements were better than 20% for concentrations below 5 ng ml(-1) and better than 10% for concentrations above 5 ng ml(-1). The method was successfully applied to plasma samples collected from a porcine pharmacokinetics study.     

Simple and efficient method for enantioselective determination of omeprazole in human plasma

A practical and selective HPLC method for the separation and quantification of omeprazole enantiomers in human plasma is presented. C18 solid phase extraction (SPE) cartridges were used to extract the enantiomers from plasma samples and the chiral separation was carried out on a Chiralpak AD column protected with a CN guard column, using ethanol:hexane (70:30) as the mobile phase, at a flow rate of 0.5 ml/min. The detection was carried out at 302 nm. The method proved to be linear in the range of 10-1000 ng/ml for each enantiomer, with a quantification limit of 5 ng/ml. Precision and accuracy, demonstrated by within-day and between-day assays, were lower than 10%.     

Gas chromatographic–mass spectrometric assay for rocuronium with potential for quantifying its metabolite, 17-desacetylrocuronium, in human plasma

A rapid, sensitive and selective method has been developed for the quantification of plasma concentrations of neuromuscular blocking drug, rocuronium, using gas chromatography with mass spectrometric detection. 3-Desacetylvecuronium served as the internal standard. The method involved iodide ion pair formation and a single-step liquid–liquid extraction with dicholoromethane. This method also permits simultaneous determination of its putative metabolite, 17-desacetylrocuronium, although the high detection limit for the metabolite limits the practical application of this method in pharmacokinetic study of the metabolite. The extraction efficiency was 75% for rocuronium and 50% for 17-desacetylrocuronium. The limit of quantification was 26 ng/ml for rocuronium and 870 ng/ml for its metabolite. The assay was used successfully in a patient undergoing liver transplantation and receiving rocuronium as a constant rate infusion and in a patient undergoing general elective surgery receiving the drug as an intravenous bolus. This assay is a time-saving alternative to published gas or liquid chromatographic methods for assaying rocuronium.     

Chromatographic and immunochemical approaches to the analysis of the HIV protease inhibitor saquinavir in plasma

The development of the HIV protease inhibitor saquinavir (Ro 31-8959) required a range of analytical methods for its measurement in biological fluids. This paper describes the development of isocratic, reverse-phase HPLC/UV methods for the routine measurement of plasma levels of the drug together with a more sensitive radioimmunoassay. The performance of the two assays is compared with that of an HPLC/MS/MS method previously published and has been shown to be satisfactory, with coefficients of variation of calibration standards and quality control samples within the usual outside limits of +/- 15%. The HPLC/UV method can be routinely applied for concentrations down to 10-20 ng/ml and a lower limit of quantification of 1 ng/ml from 1 ml of human plasma is possible. The radioimmunoassay was developed for the specific measurement of saquinavir concentrations in human, HIV-positive plasma samples and has a lower limit of quantification of 0.5-1.0 ng/ml. Some preliminary findings suggested that it might not be specific in rat plasma and no attempts have been made to quantify any nonclinical samples with this technique. If still greater sensitivity is required, recourse can be made to the HPLC/MS/MS assay.     

Simple high-performance liquid chromatographic method for the determination of ranitidine in human plasma

A simple high-performance liquid chromatographic method was developed for the determination of ranitidine in human plasma. Prior to analysis, ranitidine and the internal standard (metoprolol) were extracted from alkalinized plasma samples using dichloromethane. The mobile phase was 0.05 M potassium dihydrogenphosphate–acetonitrile (88:12, v/v) adjusted to pH 6.5. Analysis was run at a flow-rate of 1.3 ml/min and at a detection wavelength of 229 nm. The method is sensitive with a detection limit of 1 ng/ml at a signal-to-noise ratio of 3:1, while the quantification limit was set at 15 ng/ml. The calibration curve was linear over a concentration range of 15–2000 ng/ml. Mean recovery value of the extraction procedure was about 90%, while the within-day and between-day coefficients of variation and percent error values of the assay method were all less than 15%.     

Determination of lidocaine and its two N-desethylated metabolites in dog and horse plasma by high-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry

A sensitive method for the quantification of lidocaine and its metabolites, monoethylglycinexylidide (MEGX) and glycinexylidide (GX), in animal plasma using high-performance liquid chromatography combined with electrospray ionization mass spectrometry is described. The sample preparation includes a liquid-liquid extraction with methyl tert-butylmethyl ether after addition of 2M sodium hydroxide. Ethylmethylglycinexylidide (EMGX) is used as an internal standard. For chromatographic separation, an ODS Hypersil column was used. Isocratic elution was achieved with 0.01 M ammonium acetate and acetonitrile as mobile phases. Good linearity was observed in the range of 2.5-1000 ng ml(-1) for lidocaine in both dog and horse plasma. For MEGX, linear calibration curves were obtained in the range of 5-1000 ng ml(-1) and 20-1000 ng ml(-1) for dog and horse plasma, respectively. In dog and horse plasma good linearity was observed in the range of 200-1500 ng ml(-1) for GX. The limit of quantification (LOQ) in dog plasma for lidocaine, MEGX and GX was set at 2.5 ng ml(-1), 20 ng ml(-1) and 200 ng ml(-1), respectively. For horse plasma a limit of quantification of 2.5 ng ml(-1), 5 ng ml(-1) and 200 ng ml(-1) was achieved for lidocaine, MEGX and GX, respectively. In dog plasma, the limit of detection (LOD) was found to be 0.8 ng ml(-1), 2.3 ng ml(-1) and 55 ng ml(-1) for lidocaine, MEGX and GX, respectively. In horse plasma the LOD's found for lidocaine, MEGX and GX, were 1.1 ng ml(-1), 0.5 ng ml(-1) and 13 ng ml(-1), respectively. The method was shown to be of use in pharmacokinetic studies after application of a transdermal patch in dogs and after an intravenous infusion in horses.     

Stereoselective determination of cisapride, a prokinetic agent, in human plasma by chiral high-performance liquid chromatography with ultraviolet detection: application to pharmacokinetic study

We have developed a simple, sensitive, specific and reproducible stereoselective high-performance liquid chromatography technique for analytical separation of cisapride enantiomers and measurement of cisapride enantiomers in human plasma. A chiral analytical column (ChiralCel OJ) was used with a mobile phase consisting of ethanol–hexane–diethylamine (35:64.5:0.5, v/v/v). This assay method was linear over a range of concentrations (5–125 ng/ml) of each enantiomer. The limit of quantification was 5 ng/ml in human plasma for both cisapride enantiomers, while the limit of detection was 1 ng/ml. Intra- and inter-day C.V.s did not exceed 15% for all concentrations except at 12.5 ng/ml for EII (+)-cisapride, which was 20 and 19%, respectively. The clinical utility of the method was demonstrated in a pharmacokinetic study of normal volunteers who received a 20 mg single oral dose of racemic cisapride. The preliminary pharmacokinetic data obtained using the method we describe here provide evidence for the first time that cisapride exhibits stereoselective disposition.     

Determination of etoricoxib in human plasma by liquid chromatography-tandem mass spectrometry with electrospray ionisation

The validation of a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the determination of the selective cyclooxygenase-2 inhibitor etoricoxib in human plasma with phenazone as internal standard is described. The plasma samples were extracted by solid-phase extraction using polymer-based cartridges. Chromatography was carried out on a short, narrow bore RP C(18) column (30x2 mm). Detection was achieved by a Sciex API 3000 triple quadrupole mass spectrometer equipped with a turbo ion spray source working in positive ion mode. The respective mass transitions used for quantification of etoricoxib and phenazone were m/z 359.2-->280.2 and m/z 189.0-->104.1. The analytical method was validated over the concentration range 0.2-200 ng/ml. The limit of quantification was 0.2 ng/ml. The method is applicable to pharmacokinetic studies in humans.     

Determination of isosorbide-5-mononitrate in human plasma by high-resolution gas chromatography

A method for the determination of isosorbide-5-mononitrate (5-ISMN) in human plasma by capillary gas chromatography with electron-capture detection was developed and applied to clinical samples. 9-Fluorenone was used as an internal standard, ethyl acetate was employed for liquid-liquid extraction. The advantage of the extraction procedure is the possibility of a direct injection of the plasma extract, without solvent removal/reconstitution of the sample. The precision and accuracy of the method were satisfactory in the concentration range 10-1600 ng/ml. The lower limit of quantification was 10 ng/ml.