Determination of 8-oxoguanine in human plasma and urine by high-performance liquid chromatography with electrochemical detection

A highly sensitive and selective method for determining 8-oxoguanine in plasma and urine was developed by high-performance liquid chromatography with electrochemical detection. The compound was separated by gradient elution on a C₁₈ reversed-phase column with a mobile phase of acetonitrile and 0.1 M sodium acetate, pH 5.2. 8-Hydroxy-2′-deoxyguanosine was used as internal standard. 8-Oxoguanine was detected electrochemically by setting the potential to +300 mV vs. Pd reference. The sensitivity of the assay was 22 ng/ml with a signal-to-noise ratio of 7:1. The within-day relative standard deviations for 8-oxoguanine quality control samples with concentrations of 3340, 1340 and 84 ng/ml were 3.6, 4.3 and 5.7% for plasma, and 4.1, 4.6 and 6.2% for urine, respectively. The day-to-day relative standard deviations for the same samples were 3.8, 6.8 and 7.1% for plasma, and 3.9, 7.0 and 7.9% for urine, respectively. The method is designed to study the pharmacokinetics and metabolic fate of O⁶-benzylguanine in a phase I clinical trial. Previously, O⁶-benzyl-8-oxoguanine was identified as the primary metabolite of O⁶-benzylguanine in humans. We now demonstrate that 8-oxoguanine is a further metabolite of O⁶-benzylguanine.     

A high-performance liquid chromatography assay with ultraviolet detection for olanzapine in human plasma and urine

Olanzapine is a commonly used atypical antipsychotic medication for which therapeutic drug monitoring has been proposed as clinically useful. A sensitive method was developed for the determination of olanzapine concentrations in plasma and urine by high-performance liquid chromatography with low-wavelength ultraviolet absorption detection (214 nm). A single-step liquid–liquid extraction procedure using heptane-iso-amyl alcohol (97.5:2.5 v/v) was employed to recover olanzapine and the internal standard (a 2-ethylated olanzapine derivative) from the biological matrices which were adjusted to pH 10 with 1 M carbonate buffer. Detector response was linear from 1–5000 ng (r²>0.98). The limit of detection of the assay (signal:noise=3:1) and the lower limit of quantitation were 0.75 ng and 1 ng/ml of olanzapine, respectively. Interday variation for olanzapine 50 ng/ml in plasma and urine was 5.2% and 7.1% (n=5), respectively, and 9.5 and 12.3% at 1 ng/ml (n=5). Intraday variation for olanzapine 50 ng/ml in plasma and urine was 8.1% and 9.6% (n=15), respectively, and 14.2 and 17.1% at 1 ng/ml (n=15). The recoveries of olanzapine (50 ng/ml) and the internal standard were 83±6 and 92±6% in plasma, respectively, and 79±7 and 89±7% in urine, respectively. Accuracy was 96% and 93% at 50 and 1 ng/ml, respectively. The applicability of the assay was demonstrated by determining plasma concentrations of olanzapine in a healthy male volunteer for 48 h following a single oral dose of 5 mg olanzapine. This method is suitable for studying olanzapine disposition in single or multiple-dose pharmacokinetic studies.     

Determination of free concentrations of ropivacaine and bupivacaine in plasma from neonates using small-scale equilibrium–dialysis followed by liquid chromatography-mass spectrometry

Attempts to determine a safe plasma concentration of ropivacaine and bupivacaine in neonates have not been consistent. This might be due to an underestimation of free drug in small plasma samples by currently used techniques, e.g., ultrafiltration. We describe a simple microscale equilibrium–dialysis technique for the separation of free and bound ropivacaine and bupivacaine. The free drug in the dialysate was determined using solid-phase extraction and liquid chromatography with mass spectrometry. Pentycaine was used as an internal standard and added to the dialysates prior to extraction. The method is very selective and sensitive, as no compounds other than the analyte and internal standard were observed in the resulting chromatograms at low ng/ml levels. The limit of quantitation was 2.5 ng/ml. The calibration curve was linear in the range of 2 to 1000 ng/ml. The precision of the whole procedure was 8.1% (n=10) and 6.5% (n=7) for ropivacaine and bupivacaine, respectively. The method was tested in the analysis of plasma samples taken from neonates who had received epidural injections.     

Development of a gradient elution high-performance liquid chromatography assay with ultraviolet detection for the determination in plasma of the anticancer peptide [Arg, -Trp, mePhe]-substance P (6–11) (antagonist G), its major metabolites and a C-terminal pyrene-labelled conjugate

[Arg⁶, -Trp^7,9, mePhe⁸]-substance P (6–11), code-named antagonist G, is a novel peptide currently undergoing early clinical trials as an anticancer drug. A sensitive, high efficiency high-performance liquid chromatography (HPLC) method is described for the determination in human plasma of antagonist G and its three major metabolites, deamidated-G (M1), G-minus Met¹¹ (M2) and G[Met¹¹(O)] (M3). Gradient elution was employed using 40 mM ammonium acetate in 0.15% trifluoroacetic acid as buffer A and acetonitrile as solvent B, with a linear gradient increasing from 30 to 100% B over 15 min, together with a microbore analytical column (μBondapak C₁₈, 30 cm×2 mm I.D.). Detection was by UV at 280 nm and the column was maintained at 40°C. Retention times varied by <1% throughout the day and were as follows: G, 13.0 min; M1, 12.2 min; M2, 11.2 min; M3, 10.8 min, and 18.1 min for a pyrene conjugate of G (G–P). The limit of detection on column (LOD) was 2.5 ng for antagonist G, M1–3 and G–P and the limit of quantitation (LOQ) was 20 ng/ml for G and 100 ng/ml for M1–3. Sample clean-up by solid-phase extraction using C₂-bonded 40 μm silica particles (Bond Elut, 1 ml reservoirs) resulted in elimination of interference from plasma constituents. Within-day and between-day precision and accuracy over a broad range of concentrations (100 ng/ml–100 μg/ml) normally varied by <10%, although at the highest concentrations of M1 and M2 studied (50 μg/ml), increased variability and reduced recovery were observed. The new assay will aid in the clinical development of antagonist G.     

Determination of idarubicin and idarubicinol in rat plasma using reversed-phase high-performance liquid chromatography and fluorescence detection

A reversed-phase high-performance liquid chromatographic method is described for the simultaneous determination of idarubicin and idarubicinol in rat plasma. Blood samples were analyzed from 16 rats which had received an intravascular dose of 2.25 mg kg⁻¹ idarubicin. After deproteinization with acetonitrile, the separation was performed with a LiChrospher 100 RP-18 column (5 μm), using fluorescence detection (excitation: 485 nm/emission: 542 nm). The mean recovery was 95.6% for idarubicin and 90.7% for idarubicinol, respectively. The detection limit was 0.25 ng ml⁻¹ using an injection volume of 50 μl. Daily relative standard deviation (RSD) was 3.2% (10 ng idarubicin/ml, n=10) and 4.4% (10 ng idarubicinol/ml, n=10).     

Determination of gacyclidine enantiomers in human plasma by gas chromatography–mass spectrometry using selected-ion monitoring

A sensitive gas chromatographic assay using mass selective-detection has been developed for the simultaneous quantitation of the enantiomers of (±)-gacyclidine (a non competitive N-methyl-

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-aspartate antagonist) in human plasma. Gacyclidine enantiomers and phencyclidine (PCP), the internal standard, were extracted using a single-step liquid–liquid extraction with hexane at pH 8.0. Each enantiomer was separated on a chiral gas chromatography capillary column and specifically detected by mass spectrometry (MS) in selected-ion monitoring (SIM) mode. Gacyclidine enantiomers and PCP were monitored using the fragment ions at m/z 206 and 200, respectively. No interference was observed from endogenous components. The limit of quantitation (LOQ) for each enantiomer of gacyclidine was 300 pg/ml by using plasma samples of 500 μl. The calibration curves were linear (r²=0.998) over a range of 0.3125 to 20 ng/ml. The extraction efficiency was higher than 95% for both enantiomers. Intra- and inter-day bias were less than 10% at every standard curve concentration. Intra-day precision was less than 19% for (−)-gacyclidine and 15% for (+)-gacyclidine. Inter-day precision was below 15% for both enantiomers. The assay was validated for an enantioselective pharmacokinetic study in healthy male volunteers.     

Direct analysis of nitrocatechol-type glucuronides in urine by capillary electrophoresis–electrospray ionisation mass spectrometry and tandem mass spectrometry

Direct, quantitative capillary electrophoresis–electrospray ionisation mass spectrometric (CE–ESI-MS) and tandem mass spectrometric (CE–ESI-MS–MS) methods are described for the quantitation of 3-O-glucuronides of E- and Z-entacapone isomers (EEG and EZG) and tolcapone (TG) in urine. 3-O-Glucuronide of nitecapone was used as internal standard. Good separation of glucuronides was achieved with 20 mM ammonium acetate as separation solution at pH 6.84. Stacking was used to increase the sensitivity of the method by introducing samples in 5 mM ammonium acetate. CE–ESI-MS and CE–ESI-MS–MS methods are linear with correlation coefficients better than 0.9983 and 0.9982, and repeatable with relative standard deviations below 9 and 14%, respectively. The limit of detection (LOD) in CE–ESI-MS at signal-to-noise ratio 3 is 100 ng/ml for EEG and EZG and 250 ng/ml for TG. The CE–ESI-MS–MS method was the more sensitive; LOD was 7 ng/ml for all compounds, without any concentration of the sample.     

Analysis of N-methylhistamine by gas chromatography–mass spectrometry

A gas chromatography–electron capture mass spectrometry assay has been developed for the histamine H₃ receptor agonist, N^α-methylhistamine (N^α-MH). The assay is linear from 50 pg–10 ng, with a limit of detection of 50 pg/ml for gastric juice and plasma, and 50 pg/sample for bacteria (10⁷–10⁸ CFU) and gastric tissue (5–10 mg wet weight). The limits of quantification are 100 pg/ml for gastric juice (%RSD=1.4) and plasma (%RSD=9.4), and 100 pg/sample for bacteria (%RSD=3.9) and tissue (%RSD=5.8). N^α-MH was not present in human plasma, but low levels (1.4 ng/ml and 0.4 ng/ml) were detected in two samples of human gastric juice obtained from patients infected with Helicobacter pylori.     

Simultaneous determination of prednisolone, prednisolone acetate and hydrocortisone in human serum by high-performance liquid chromatography

A method for the simultaneous determination of prednisolone, prednisolone acetate and hydrocortisone has been established to monitor the serum levels of these three compounds in healthy volunteers following intramuscular administration of prednisolone acetate. Serum samples of 0.75 ml were extracted with ethyl acetate after addition of the internal standard, dexamethasone. The compounds were separated using a LiChrosorb Si 60 column and detected by UV absorbance. Specificity, linearity, as well as the repeatability, intermediate-precision and accuracy of the method were established. The lower limit of quantification was 2.0 ng/ml for prednsolone (C.V. = 14.7%, N=6) and 5.0 ng/ml for prednisolone acetate (C.V. = 13.9%, N= 6 and hydrocortisone (C.V. = 11.7%, N=6). Data on the recovery of the compounds and the internal standard are provided. The results of quality control samples determined during routine analysis (n = 114) are presented. Serum levels of the compounds after intramuscular adminstration of 25 mg of prednisolone acetate are discussed.     

Urinary excretion of 19-norandrosterone of endogenous origin in man: quantitative analysis by gas chromatography–mass spectrometry

A GC–MS method, using deuterium-labelled 19-noretiocholanolone as internal standard and following an extensive LC purification prior to selected ion monitoring of the bis(trimethylsilyl) ethers at ion masses m/z 405, 419, 420 and 421, allowed the quantitation of subnanogram amounts of 19-norandrosterone present in 10-ml urine samples at m/z 405. Thirty healthy men, free of anabolic androgen supply, delivered 24-h urine collections in 4 timed fractions. Accuracy was proven by the equation, relating added (0.05–1 ng/ml) to measured analyte, which had a slope not significantly different from 1. Precision (RSD) was 4% at a concentration of 0.4 ng/ml, and 14% at 0.04 ng/ml. Analytical recovery was 82%. The limit of quantitation was 0.02 ng/ml. The excretion ranges were 0.03–0.25 μg/24 h or 0.01–0.32 ng/ml in nonfractionated 24-h urine.Taking into account inter-individual variability and log-normal distribution, a threshold of 19-norandrosterone endogenous concentration of 2 ng/ml, calculated as the geometric mean plus 4 SD, was established. This value corresponds to the decision limit advised by sport authorities for declaring positive (anabolic) doping with nandrolone.     

Preconcentration with Bacillus subtilis–Immobilized Amberlite XAD-16: Determination of Cu2+ and Ni2+ in River,Soil, and Vegetable Samples

Solid-phase extraction (SPE) method was developed for the preconcentration of Cu²⁺ and Ni²⁺ before their determination by inductively coupled plasma optical emission spectrometry (ICP-OES). Bacillus subtilis–immobilized Amberlite XAD-16 was used as biosorbent. Effects of critical parameters such as pH, flow rate of samples, amount of Amberlite XAD-16 and biosorbent, sample volume, eluent type, and volume and concentration of eluent on column preconcentration of Cu²⁺ and Ni²⁺ were optimized. Applicability of the method was validated through the analysis of the certified reference tea sample (NCS ZC73014). Sensitivity of ICP-OES was improved by 36.4-fold for Cu²⁺ and 38.0-fold for Ni²⁺ by SPE-ICP-OES method. Limit of quantitation (LOQ) was found to be 0.7 and 1.1 ng/ml for Cu²⁺ and Ni²⁺, respectively. Concentrations of Cu²⁺ and Ni²⁺ were determined by ICP-OES after application of developed method. Relative standard deviations (RSDs) were lower than 4.9% for Cu²⁺ and 7.9% for Ni²⁺. The Tigris River that irrigates a large agricultural part of Southeast Turkey is polluted by domestic and industrial wastes. Concentrations of Cu²⁺ and Ni²⁺ were determined in water, soil, and some edible vegetables as a biomonitor for heavy metal pollution.     

Sensitive and selective assay for fentanyl using gas chromatography with mass selective detection

A modified gas chromatographic assay, using mass-selective detection, has been developed for the quantitation of fentanyl in swine serum. Fentanyl and sufentanil, the internal standard, were extracted using a single-step liquid-liquid extraction with dichloromethane. Sensitivity and selectivity were improved by using electron-impact ionization (EI) in the selected-ion monitoring (SIM) mode, where fentanyl and sufentanil were monitored using the fragment ions at m/z 245 and 289, respectively. The limit of quantitation (LOQ) is 0.05 ng/ml, using 1 ml of sample, with a C.V. of 10.8% and a signal-to-noise ratio of 29. Standard curves were linear (r² = 0.999) over the working range of 0.05–1.5 ng/ml, using 1/y² as a weighting factor. Recoveries averaged 69.8 ± 4.7%, 91.0 ± 13.0% and 90.9 ± 10.3% at serum concentrations of 1.5, 0.5 and 0.1 ng/ml, respectively. Intra- and inter-day variances, were <12% at 0.1 ng/ml, and <10% at concentrations of 0.5, 1 and 1.5 ng/ml. Bias was 6.2% at the LOQ and ⩽12.8% at every other standard curve concentration. Applicability of the assay is demonstrated for the pharmacokinetic study of transdermally administered fentanyl in a postoperative swine.     

High-performance liquid chromatographic determination of vinorelbine in human plasma and blood: application to a pharmacokinetic study

A sensitive and specific high-performance liquid chromatographic method with fluorescence detection (excitation wavelength: 280 nm; emission wavelength: 360 nm) was developed and validated for the determination of vinorelbine in plasma and blood samples. The sample pretreatment procedure involved two liquid–liquid extraction steps. Vinblastine served as the internal standard. The system uses a Spherisorb cyano analytical column (250×4.6 mm I.D.) packed with 5 μm diameter particles as the stationary phase and a mobile phase of acetonitrile–80 mM ammonium acetate (50:50, v/v) adjusted to pH 2.5 with hydrochloric acid. The assay showed linearity from 1 to 100 ng/ml in plasma and from 2.5 to 100 ng/ml in blood. The limits of quantitation were 1 ng/ml and 2.5 ng/ml, respectively. Precision expressed as RSD was in the range 3.9 to 20% (limit of quantitation). Accuracy ranged from 92 to 120%. Extraction recoveries from plasma and blood averaged 101 and 75%, respectively. This method was used to follow the time course of the concentration of vinorelbine in human plasma and blood samples after a 10-min infusion period of 20 mg/m² of this drug in patients with metastatic cancer.     

Determination of pramipexole (U-98,528) in human plasma and urine by high-performance liquid chromatography with electrochemical and ultraviolet detection

A sensitive and selective high-performance liquid chromatographic (HPLC) method was developed for the determination of pramipexole in human plasma and urine. Plasma/urine is made alkaline before pramipexole and BHT-920 (internal standard) are extracted by ethyl ether and back-extracted with a solution that contains heptanesulfonic acid. Separation is achieved by ion-pair chromatography on a Zorbax Rx C₈ column with electrochemical detection at 0.6 V for plasma and ultraviolet detection at 286 nm for urine. The retention times of pramipexole and internal standard are approximately 14.4 and 10.7 min, respectively. The assay is linear in concentration ranges of 50 to 15 000 pg/ml (plasma) and 10 to 10 000 ng/ml (urine). The correlation coefficients are greater than 0.9992 for all curves. For the plasma method, the analysis of pooled quality controls (300, 3000, and 10 000 pg/ml) demonstrates excellent precision with relative standard deviations (R.S.D.) (n=18) of 1.1%, 2.3%, and 6.8%, respectively. For the urine method, quality control pools prepared at 30, 300, and 3000 ng/ml had R.S.D. values (n=18) of 2.9%, 1.7%, and 3.0%, respectively. The plasma and urine controls were stable for more than nine and three months, respectively. The mean recoveries for pramipexole and internal standard from plasma were 97.7% and 98.2%, respectively. The mean recoveries for pramipexole and internal standard from urine were 89.8% and 95.1%, respectively. The method is accurate with all intra-day (n=6) and overall (n=18) mean values for the quality control samples being less than 6.4 and 5.8% from theoretical for plasma and urine, respectively.     

Determination of dihydroetorphine in biological fluids by gas chromatography-mass spectrometry using selected-ion monitoring

A method for the determination of dihydroetorphine hydrochloride, a powerful anaesthetic and analgesic drug, in biological fluids by GC-MS with selected-ion monitoring using etorphine as internal standard was established. Dihydroetorphine was extracted from human blood and urine with dichloromethane and then derivatized with N-heptafluorobutyrylimidazole after concentration to dryness. A dihydroetorphine monoheptafluorobutyl derivative was formed which showed good behavior on GC-MS with electronic-impact ionization. The main fragment, m/z 522, which is the base peak, was selected as the ion for quantitation and the corresponding ion, m/z 520, was selected for monitoring the internal standard, etorphine. The recoveries and coefficients of variation of the whole procedure were determined with five controlled dihydroetorphine-free urine and plasma samples spiked with different concentrations of dihydroetorphine. The concentration of dihydroetorphine for quantitation was in the range 1–20 ng/ml for urine and 2.5–250 ng/ml for plasma. The correlation coefficients of the standard curves are sufficient to determine the dihydroetorphine. The accuracy for quantitation of dihydroetorphine in urine and plasma is less than 10.6%.     

Efficient assay for the determination of atenolol in human plasma and urine by high-performance liquid chromatography with fluorescence detection

An efficient method for the determination of atenolol in human plasma and urine was developed and validated. α-Hydroxymetoprolol, a compound with a similar polarity to atenolol, was used as the internal standard in the present high-performance liquid chromatographic analysis with fluorescence detection. The assay was validated for the concentration range of 2 to 5000 ng/ml in plasma and 1 to 20 μg.ml in urine. For both plasma and urine, the lower limit of detection was 1 ng/ml. The intra-day and inter-day variabilities for plasma samples at 40 and 900 ng/ml, and urine samples at 9.5 μg/ml were <3% (n=5).     

Manual and automatic extraction and high-performance liquid chromatographic determination of a spicamycin derivative, KRN5500, in rat plasma

A sensitive reliable method for the extraction, separation and quantitation of KRN5500 (I), a spicamycin derivative, from rat plasma was developed. It involves solid-phase extraction of the drug using a Bond Elut C₁₈ cartridge and reversed-phase HPLC on a YMC-Pack ODS column with an ultraviolet detector. The intra- and inter-assay coefficients of variation by manual (n=10) and automatic (n=5) extraction were less than 9 and 13% and 6 and 8%, respectively. The limit of quantitation of each extraction procedure was 2 ng potency/ml. This extraction method may thus be considered useful for monitoring I in animals following its administration.     

Determination of marbofloxacin in plasma samples by high-performance liquid chromatography using fluorescence detection

A simple and sensitive HPLC method has been developed for the determination of marbofloxacin (MAR) in plasma. Sample preparations were carried out by adding phosphate buffer (pH 7.4, 0.1 M), followed by extraction with trichloromethane. MAR and the internal standard, enrofloxacin (ENR), were separated on a reversed-phase column and eluted with aqueous solution–acetonitrile (80:20). The fluorescence of the column effluent was monitored at λ_ex=338 and λ_em=425 nm. The retention times were 2.20 and 3.30 min for MAR and ENR, respectively. The method was shown to be linear from 15 to 1500 ng/ml (r²=0.999). The detection limit was 15 ng/ml. Mean recovery was determined as 90% by the analysis of plasma standards containing 150, 750, and 1500 ng/ml. Inter- and intra-assay precisions were 3.3% and 2.7%, respectively.     

Monitoring the gestation period of rescued Formosan pangolin (Manis pentadactyla pentadactyla) with progesterone radioimmunoassay

Eight species of pangolin have been identified in the world. However, understanding of pangolin reproductive biology has been limited to fragmentary records. In this study, the concentration of serum progesterone in three pregnant and two nonpregnant rescued female Formosan pangolins (Manis pentadactyla pentadactyla) was monitored using a commercial progesterone radioimmunoassay kit. During gestation, the serum progesterone of pregnant pangolins A, B, and C remained at 28.5–55 ng/ml (n = 31 samples), 10.9–50.1 ng/ml (n = 34), and 12.4 and 33.5 ng/ml with a peak at 47.6 ng/ml (n = 19), respectively, whereas the serum progesterone of nonpregnant pangolins D and E remained at 1.99 ± 1.62 ng/ml (n = 80) and 2.27 ± 1.64 ng/ml (n = 27), respectively. From this study, it was found that female pangolin weighing as low as 2.14 kg was already capable of reproduction. For pregnant pangolins to give birth to viable offspring, their body weight must increase significantly, 63.89 and 134.0% in the study, from the time of inception or early pregnancy until parturition. In addition, study has found that both viable offspring were born fully developed and exceeded 80 g in weight. The period of gestation was found to be as short as 318 or longer than 372 days. Therefore, the Formosan pangolin should only be able to reproduce once a year. This study is the first insight into hormone assay for determining the gestation period of pangolin. Further investigations on the same subject are necessary to establish criteria for the recognition of reproductive status in pangolins. Zoo Biol 31:479–489, 2012. © 2011 Wiley Periodicals, Inc.     

Determination of calcium dobesilate in human plasma using ion-pairing extraction and high-performance liquid chromatography

A rapid, simple reversed-phase high-performance liquid chromatographic method with ultraviolet absorbance detection has been developed for the determination of calcium dobesilate in human plasma. Sample processing is based on an ion-pairing extraction with tetra-n-butylammonium hydroxide as cationic pairing ion and dichloromethane. Separation of the investigated calcium dobesilate and 2,4-dihydroxybenzoic acid as internal standard was achieved on a Discovery RP-Amide C₁₆ analytical column with 50 mM, pH 2.5, potassium dihydrogenphosphate buffer–acetonitrile (75:25, v/v) mobile phase. The wavelength was set at 305 nm. The limit of quantitation is 100 ng/ml and the calibration curve is linear up to 50 μg/ml. Within-day and between-day precision expressed as the relative standard deviation is about 10% and the accuracy of the determination did not deviate from 100% by more than ±10%. The developed method was found to be suitable for application in human bioequivalence studies.