Simultaneous determination of oxycodone and its major metabolite, noroxycodone, in human plasma by high-performance liquid chromatography

Oxycodone (14-hydroxy-7,8-dihydrocodeinone) is a potent opioid receptor agonist. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with UV detection was validated and applied for the analysis of oxycodone and its major metabolite, noroxycodone, in human plasma. The analytes were separated using a mobile phase, consisting of acetonitrile and phosphate buffer (8:92, v/v) at a flow rate of 1 mL/min, and UV detection at 205 nm. The retention times for oxycodone, noroxycodone and codein (internal standard) were 14.7, 13.8 and 10.2 min, respectively. The validated quantitation range of the method was 2-100 ng/mL for oxycodone and 10-100 ng/mL for noroxycodone. The developed procedure was applied to assess the pharmacokinetics of oxycodone and its metabolite following administration of a single 20 mg oral dose of oxycodone hydrochloride to one healthy male volunteer.     

The use of liquid chromatography/mass spectrometry for quantitative analysis of oxycodone, oxymorphone and noroxycodone in Ringer solution, rat plasma and rat brain tissue

Sensitive and reproducible methods for the determination of oxycodone, oxymorphone and noroxycodone in Ringer solution, rat plasma and rat brain tissue by liquid chromatography/mass spectrometry are described. Deuterated analogs of the substances were used as internal standards. Samples in Ringer solution were analyzed by direct injection of 10 microL Ringer solution diluted by an equal volume of water. The limit of quantification was 0.5 ng/mL and the method was linear in the range of 0.5-150 ng/mL for all substances. To analyze oxycodone and oxymorphone in rat plasma, 50 microL of plasma were precipitated with acetonitrile, and the supernatant was directly injected onto the column. To analyze oxycodone, oxymorphone and noroxycodone in rat plasma, 100 microL of rat plasma were subjected to a C18 solid-phase extraction (SPE) procedure, before reconstituting in mobile phase and injection onto the column. For both methods the limit of quantification in rat plasma was 0.5 ng/mL and the methods were linear in the range of 0.5-250 ng/mL for all substances. To analyze the content of oxycodone, oxymorphone and noroxycodone in rat brain tissue, 100 microL of the brain homogenate supernatant were subjected to a C18 SPE procedure. The limit of quantification of oxycodone was 20 ng/g brain, and for oxymorphone and noroxycodone 4 ng/g brain, and the method was linear in the range of 20-1000 ng/g brain for oxycodone and 4-1000 ng/g brain for oxymorphone and noroxycodone. All methods utilized a mobile phase of 5 mM ammonium acetate in 45% acetonitrile, and a SB-CN column was used for separation. The total run time of all methods was 9 min. The intra-day precision and accuracy were <11.3% and <+/-14.9%, respectively, and the inter-day precision and accuracy were <14.9% and <+/-6.5%, respectively, for all the concentrations and matrices described.     

A rapid and sensitive high-performance liquid chromatography-electrospray ionization-triple quadrupole mass spectrometry method for the quantitation of oxycodone in human plasma

A sensitive method for the determination of oxycodone concentrations in plasma by high-performance liquid chromatography (HPLC)-electrospray ionization-triple quadrupole mass spectrometry is described. The method is rugged, reliable, selective, and rapid with a run time of 2 min. One milliliter of plasma is made basic and extracted with 2-mL duplicate portions of 2% isoamyl alcohol in n-butyl chloride. The combined extracts are then evaporated to dryness, reconstituted in 100 microL of the mobile phase (15% methanol-85% water containing 0.1% acetic acid), and injected onto the HPLC. The limit of quantitation is 1 ng/mL, and the estimated limit of detection is 33 pg/mL (signal-to-noise = 3). Standard curves are linear over the range of 1 to 100 ng/mL with all correlation coefficient values greater than 0.9989. The method is used to determine the concentration of oxycodone in human plasma following the intravenous infusion of doses ranging from 5 to 15 mg in which the analysis of over 3000 plasma samples is required.     

Determination of four lignans in Phyllanthus niruri L. by a simple high-performance liquid chromatography method with fluorescence detection

A new and simple analytical method using HPLC with fluorescence detection was developed for the simultaneous determination of four lignans (phyllanthin, hypophyllanthin, phyltetralin and niranthin) in Phyllanthus niruri L. plant samples. Optimal separation was achieved with an isocratic mobile phase consisting of acetonitrile-water (55:45 v/v). The method recorded limits of detection (S/N=5) for phyllanthin at 0.61 ng/mL, hypophyllanthin at 6.02 ng/mL, phyltetralin at 0.61 ng/mL and niranthin at 1.22 ng/mL, being 80, 8, 80 and 40 times, respectively, lower when compared with those derived using HPLC-UV detection. The limits of quantification (S/N=12) were 4.88 ng/mL for phyllanthin and phyltetralin, 9.76 ng/mL for niranthin and 24.4 ng/mL for hypophyllanthin showing 40, 8 and 20 times, respectively, lower than those from the UV detection method. The within-day and between-day accuracy for the four lignans were between 98.1% and 102.9% while their precision values were below 2.2%. The mean recovery was between 92.5% and 110.1%. The method was then successfully applied for the quantification of lignans in P. niruri plant samples. The highest amount of lignans was found in the leaves followed by fruits, branches and stem, whilst the roots have the least amount of lignans.     

Use of gas chromatography/mass spectrometry with positive chemical ionization for the determination of opiates in human oral fluid

An analytical method for the simultaneous determination of codeine, morphine and 6-acetylmorphine (6AM) in human oral fluid was developed. The method involves liquid-liquid extraction in Toxitubes A, derivatization with 99:1 (v/v) N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)/trimethylchlorosilane (TMCS), and gas chromatography/mass spectrometry with positive chemical ionization (GC/PCI-MS) determination. The detector response was linear over the concentration range 30-500 ng/mL with coefficients of correlation higher than 0.99. The precision was acceptable with coefficients of variation less than 7.5%. The limits of detection achieved were 0.7 ng/mL for codeine, 2.0 ng/mL for morphine, and 0.6 ng/mL for 6AM. The method proposed was applied to 80 oral fluid samples from opiates users, 98% of which were positive for the three analytes. Human oral fluid is a suitable biological fluid for the determination of opiates by GC/PCI-MS.     

New HPLC-MS method for quantitative determination of apovincaminic acid in human plasma

A fast and sensitive HPLC-MS method for the quantification of apovincaminic acid, a vinpocetine metabolite, in human plasma has been developed and validated. After protein precipitation with methanol, 10 mL of supernatant was injected at 45 degrees C onto a Zorbax SB-C18 column. Elution was performed in less than three minutes with water containing 0.2% formic acid and acetonitrile (80:20) at 0.75 mL/min. A linearity domain between 4 and 240 ng/mL and a limit of quantification of 4 ng/mL apovincaminic acid were established by monitoring the signal corresponding to m/z = 323. Accuracy and precision were less than 5.2% for within-run assay and 10% for between-run assay. The method was successfully applied in a bioequivalence study of two pharmaceutical products containing 5 mg vinpocetine.     

Preparation of a Monoclonal Antibody against a Kallikrein-Like Enzyme from Agkistrodon halys pallas Venom and Its Application in a Pharmacokinetic Study

Snake venom contains bioactive materials for drug development, diagnosis, and treatment. After separating and purifying the kallikrein-like enzyme (AHP-Ka) from Agkistrodon halys pallas venom for the first time, a monoclonal antibody against AHP-Ka was prepared and characterized. An indirect sandwich enzyme-linked immunosorbent assay (ELISA) based on the monoclonal antibody was developed and validated for the pharmacokinetic analysis of AHP-Ka in rat plasma. The method was calibrated using rat plasma and 1:100 dilution of plasma was selected to prepare a calibration curve to validate the precision, accuracy, and stability of the ELISA method. A good linear relationship was obtained in a working range from 3.9 ng/mL to 62.5 ng/mL with a limit of detection of 2.94 ng/mL. Intra- and inter-batch precision were less than 10%. The average recovery ranged from 94.6% to 104.4% in rat plasma at the concentrations of 5 ng/mL, 15 ng/mL, and 45 ng/mL, respectively. The ELISA method was successfully used for the pharmacokinetic study of AHP-Ka in Sprague-Dawley rat plasma after intravenous administration. The work is expected to contribute to future preclinical development of AHP-Ka.     

Determination of the antibacterial drug sulfamethoxazole in pharmaceutical preparations containing trimethoprim by spectrofluorimetry after derivatization with fluorescamine

An analytical method was developed for the determination of sulfamethoxazole (SMZ) in the presence of trimethoprim (TMP) by normal fluorescence. When both analytes are present a selective derivatization with fluorescamine of SMZ gives an intense fluorescent derivative with no interference from the other compound. The reaction was optimized to obtain the best analytical performance. The detection limit and the lower limit of quantitation of SMZ in the reaction medium was 5.2 ng/mL. The intra-day precision (relative standard deviation) was 1.51% for a 100 ng/mL SMZ standard solution and the inter-day precision over 7 days for a 100 ng/mL solution in the presence of 20 ng/mL TMP solution was 2.5%. The method has been applied to three pharmaceutical preparations containing both compounds, without any separation steps required. Received: 28 December 1998 / Revised: 28 April 1999 / Accepted: 30 April 1999     

A rapid liquid chromatography-tandem mass spectrometry method for quantification of a biologically active molecule camboginol in the extract of Garcinia cambogia

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of camboginol in the extract of fruit rinds of Garcinia cambogia has been developed. Separation was achieved isocratically on an RP C(18) column using a solvent system consisting of a mixture of acetonitrile-water (9:1) and methanol-acetic acid (99.5:0.5) in the ratio of 30:70 as mobile phase at a flow rate of 0.4 mL/min. A multiple reaction monitoring (MRM) method was developed for quantification of camboginol in the fruit rinds extract of G. cambogia using MRM transitions of m/z 601.4 --> m/z 176.7 and m/z 601.4 --> m/z 448.9, respectively. The calibration curve based on peak area against concentration was linear up to 50 ng/mL with a detection limit of 0.5 ng/mL. The method showed satisfactory reproducibility with a coefficient of variation of less than 6%. The method was successfully applied for quantification of camboginol in different Garcinia extracts.     

Identification and quantification of 34 drugs and toxic compounds in blood,urine, and gastric content using liquid chromatography with tandem mass spectrometry

A liquid chromatography with tandem mass spectrometry method was developed for the simultaneous screening of 34 drugs and poisons in forensic cases. Blood (0.5 mL, diluted 1:1 with water) or 1.0 mL of urine was purified by solid‐phase extraction. Gastric contents (diluted 1:1 with water) were treated with acetonitrile, centrifuged, and supernatant injected. Detection was achieved using a Waters Alliance 2695/Quattro Premier XE liquid chromatography tandem mass spectrometry system equipped with electrospray ionization, operated in the multiple reaction monitoring modes. The method was validated for accuracy, precision, linearity, and recovery. The absolute recovery of drugs and toxic compounds in blood was greater than 51% with the limit of detection in the range of 0.02–20 ng/mL. The absolute recovery of drugs and toxic compounds in urine was greater than 61% with limit of detection in the range of 0.01–10 ng/mL. The matrix effect of drugs and toxic compounds in urine was 65–117% and 67–121% in blood. The limit of detection of drugs and toxic compounds in gastric content samples were in the range of 0.05–20 ng/mL. This method was applied to the routine analysis of drugs and toxic compounds in postmortem blood, urine, and gastric content samples. The method was applied to actual forensic cases with examples given.     

Determination of vitexin and isovitexin in pigeonpea using ultrasonic extraction followed by LC-MS

A method based on ultrasonic extraction (USE) followed by LC-MS is presented for the determination of vitexin and isovitexin in pigeonpea extracts in this study. The influential parameters of the USE procedure were optimized, and the optimal conditions were as follows: extraction solvent, 60% ethanol solution; liquid/solid ratio 10:1 (mL/g), extraction power, 250 W; temperature, 40-50 degrees C; and three extraction cycles, each cycle 15 min. Validation of the USE method was performed in terms of repeatability and reproducibility. RSDs for extraction yields were lower than 5.85 and 8.09%, respectively. The LOD and LOQ of chromatographic determination were 0.96 and 3.2 ng/mL for vitexin and 0.84 and 2.8 ng/mL for isovitexin. The method was also successfully applied for the determination of vitexin and isovitexin in stems, leaves, and root extracts of pigeonpea. From all these results, we may conclude that the developed method is appropriate for the quality control of pigeonpea and other plant extract products developed from pigeonpea.     

Evaluation of an Element-Tagged Duplex Immunoassay Coupled with Inductively Coupled Plasma Mass Spectrometry Detection: A Further Study for the Application of the New Assay in Clinical Laboratory

Background: Element-tagged immunoassay coupled with inductively coupled plasma mass spectrometry (ICP-MS) detection has the potential to revolutionize immunoassay analysis for multiplex detection. However, a further study referring to the standard evaluation and clinical sample verification is needed to ensure its reliability for simultaneous analysis in clinical laboratories. Methods: Carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) were chosen for the duplex immunoassay. The performance of the assay was evaluated according to guidelines from the Clinical and Laboratory Standards Institute (CLSI). Moreover, reference intervals (RIs) of CEA and AFP were established. At last, 329 clinical samples were analyzed by the proposed method and results were compared with those obtained with electrochemiluminescent immunoassay (ECLIA) method. Results: The measurement range of the assay was 2–940 ng/mL for CEA and 1.5–1000 ng/mL for AFP, with a detection limit of 0.94 ng/mL and 0.34 ng/mL, respectively. The inter-assay and intra-assay imprecision were all less than 6.58% and 10.62%, respectively. The RI of CEA and AFP was 0–3.84 ng/mL and 0–9.94 ng/mL, respectively. Regarding to clinical sample detection, no significant difference was observed between the proposed duplex assay and the ECLIA method. Conclusions: The ICP-MS-based duplex immunoassay was successfully developed and the analytical performance fully proved clinical applicability. Well, this could be different with other analytes.     

HPLC-F analysis of melatonin and resveratrol isomers in wine using an SPE procedure

An original analytical method has been developed for the determination of the antioxidants trans-resveratrol (t-RSV) and cis-resveratrol (c-RSV) and of melatonin (MLT) in red and white wine. The method is based on HPLC coupled to fluorescence detection. Separation was obtained by using a RP column (C8, 150 mm x 4.6 mm id, 5 mum) and a mobile phase composed of 79% aqueous phosphate buffer at pH 3.0 and 21% ACN. Fluorescence intensity was monitored at lambda = 386 nm while exciting at lambda = 298 nm, mirtazapine was used as the internal standard. A careful pretreatment of wine samples was developed, using SPE with C18 cartridges (100 mg, 1 mL). The calibration curves were linear over the following concentration ranges: 0.03-5.00 ng/mL for MLT, 3-500 ng/mL for t-RSV and 1-150 ng/mL for c-RSV. The LOD values were 0.01 ng/mL for MLT, 1 ng/mL for t-RSV and 0.3 ng/mL for c-RSV. Precision data, as well as extraction yield and sample purification results, were satisfactory. Thus, the method seems to be suitable for the analysis of MLT and resveratrol isomers in wine samples. Moreover, wine total polyphenol content and antioxidant activity were evaluated.     

Simultaneous determination of niflumic acid and its prodrug, talniflumate in human plasma by high performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of niflumic acid and its prodrug, talniflumate, in human plasma. Niflumic acid and talniflumate were eluted isocratically with methanol-water (73:27, v/v, adjusted to pH 3.5 by acetic acid) at a fl ow rate of 1 mL/min. Indomethacin was used as an internal standard. Signals were monitored by an UV detector at 288 nm. Retention times of indomethacin, niflumic acid and talniflumate were 5.9, 7.2 and 13.5 min, respectively. Calibration plots were linear over the range 50-5000 ng/mL for niflumic acid and 100-5000 ng/mL for talniflumate. The limits of quantitation were 50 ng/mL for niflumic acid and 100 ng/mL for talniflumate. The intra- and inter-day relative standard deviations (RSD) of niflumic acid and talniflumate were less than 10% and the accuracies were higher than 90%. This method is rapid, sensitive and reproducible for the determination of niflumic acid and talniflumate in human plasma.     

A new assay based on solid-phase extraction procedure with LC-MS to measure plasmatic concentrations of tenofovir and emtricitabine in HIV infected patients

A new solid-phase extraction (SPE) method has been developed and validated on a liquid chromatography (LC) coupled with a mass spectrometer for the determination of plasma concentrations of tenofovir (TNF) and emtricitabine (FTC) in HIV infected patients. Chromatographic separation was achieved with a gradient (acetonitrile and water with formic acid 0.05%) on an Atlantis 4.6 mm x 150 mm, reversed phase analytical column. Detection of TNF, FTC, and internal standard (IS) was achieved by electrospray ionization mass spectrometry (ESI-MS) in the positive ion mode. Calibration ranged from 15.6 to 4000 ng/mL for TNF and 11.7 to 3000 ng/mL for FTC. Plasma was analyzed, and the limit of quantitation was 15.6 ng/mL for TNF and 11.7 ng/mL for FTC; limit of detection was 2 ng/mL for TNF and 1.5 ng/mL for FTC. Mean recovery of TNF, FTC, and IS were 46.5% [relative standard deviation (RSD): 8.8%] and 88.8% (RSD: 1.0%), and 81.7% (RSD: 3.1%), respectively. The method did not show any significant interference with antiretrovirals or other concomitant drugs administered to patients, and no significant "matrix effects" were observed. The method was applied for the determination of antiretroviral plasma concentration of HIV-positive patients treated with FTC and/or TNF, in combination with various other antiretrovirals.     

毛细管电泳-电致化学发光法测定兔血浆中的羟考酮

基于稀土Eu(Ⅲ)掺杂的类普鲁士蓝膜修饰的铂电极为工作电极,建立了测定羟考酮的毛细管电泳-电致化学发光分析方法。考察了检测电位、运行缓冲溶液的酸度及浓度、分离电压、进样条件等对电泳分离效果及检测灵敏度的影响。在最佳的实验条件下,羟考酮可在4 min内得到分离,其ECL强度值与羟考酮的质量浓度在7.0×10-2～7.0μg/mL和7.0～70.0μg/mL范围内呈良好的线性关系,检出限为4.2×10-2μg/mL(3σ),峰高和迁移时间的相对偏差分别为3.6%和0.48%(n=6)。方法用于兔血浆中羟考酮含量的检测,加标回收率在99.7%～101.0%之间。     

Extraction of clenbuterol from calf urine using a molecularly imprinted polymer followed by quantitation by high-performance liquid chromatography with UV detection

A method for the extraction of clenbuterol from calf urine samples using a molecularly imprinted polymer (MIP) has been developed. The aim was that the final extracts from the MIP should allow quantitation of clenbuterol down to 0.5 ng/mL urine using HPLC with UV detection. The MIP was produced using brombuterol as a template and the selectivity of the MIP, for clenbuterol, was tested against a non-imprinted polymer (produced without template) and was found to be high. After loading of 5 mL diluted centrifuged urine, selective binding was established in acetonitrile-acetic acid (98:2). For further elution of interferences, 0.5 M ammonium acetate buffer pH 5 and 70% acetonitrile in water was used. Clenbuterol was eluted using 1% trifluoroacetic acid in methanol, which was evaporated and reconstituted in buffer. Results from the HPLC analyses showed that the extraction of clenbuterol using MIP is linear in the range 0.5-100 ng/mL with good precision (4.3% for 0.6 ng/mL and 2.1% for 6.0 ng/mL) and accuracy (96.7% for 0.6 ng/mL and 96.7% for 6.0 ng/mL). The recoveries were 75%. The results show that the method offers a selectivity and sensitivity that make the quantitation of 0.5 ng clenbuterol/mL urine by HPLC-UV possible and a competitive alternative to state-of-the-art routine analytical methods.     

Simultaneous determination of rabeprazole and its two active metabolites in human urine by liquid chromatography with tandem mass spectrometry and its application in a urinary excretion study

A simple and rapid liquid chromatography with tandem mass spectrometry method has been developed and validated for the determination of rabeprazole and its two active metabolites, rabeprazole thioether and desmethyl rabeprazole thioether, in human urine using donepezil as the internal standard. The sample preparation procedure involved a simple dilution of urine sample with methanol (1:3, v/v). The chromatographic separation was achieved on a Hedera ODS‐2 C₁₈ column using a mixture of methanol/10 mmol/L ammonium acetate solution (containing 0.05% formic acid; 55:45, v/v) as the mobile phase. The method was validated over the concentration ranges of 0.15–100 ng/mL for rabeprazole, 0.30–400 ng/mL for rabeprazole thioether, and 0.05–100 ng/mL for desmethyl rabeprazole thioether. The established method was highly sensitive with a lower limit of quantification of 0.15 ng/mL for rabeprazole, 0.30 ng/mL for rabeprazole thioether, and 0.05 ng/mL for desmethyl rabeprazole thioether. The intra‐ and interbatch precision was <4.5% for the low, medium, and high quality control samples of all the analytes. The recovery of the analytes was in the range 95.4–99.0%. The method was successfully applied to a urinary excretion profiles after intravenous infusion administration of 20 mg rabeprazole sodium in healthy volunteers.     

Development of a LC-ESI/MS/MS assay for the quantification of vanillin using a simple off-line dansyl chloride derivatization reaction to enhance signal intensity

Vanillin is responsible for producing the familiar smell of vanilla. Vanillin has many similarities with other flavor phenolic compounds and could potentially show similar pharmacological activity. A previously published analytical method was adapted, developed and tested. Vanillin was extracted from rat plasma using protein precipitation with acetone. Prior to LC-ESI/MS/MS analysis, an aliquot of the supernatant was used to proceed to the derivatization of vanillin and the internal standard with dansyl chloride to enhance signal intensity in positive electrospray mode. The chromatography was performed on a 100 x 2.1 mm C8 column and an isocratic mobile phase composed of 75:25 acetonitrile:0.5% formic acid in water with a flow rate fixed at 500 microL/min. A linear (weighted 1/concentration) relationship was used to perform the calibration over an analytical range of 10-10,000 ng/mL. The intra-batch precision and accuracy at the limit of quantitation (10 ng/mL), medium (500 ng/mL) and high (10,000 ng/mL) concentrations were 10.7, 7.0 and 7.2% and 103.5, 108.0 and 100.1%, respectively. The observed recovery was greater than 87% and no significant ionization suppression or matrix effect was observed. This LC-ESI/MS/MS method for the determination of vanillin in rat plasma provided results within generally accepted criteria used for bioanalytical assay.     

Simultaneous determination and pharmacokinetic studies of aloe emodin and chrysophanol in rats after oral administration of Da-Cheng-Qi decoction by high-performance liquid chromatography

A validated high-performance liquid chromatography (HPLC) method was developed for simultaneous determination and pharmacokinetic study of aloe emodin and chrysophanol in rats. It was performed on a reverse-phase C(18) column and a mobile phase made up of methanol and 0.2% acetic acid (83:17, v/v). The ultraviolet detection was 254 nm. 1,8-dihydroxyanthraquinone was used as the internal standard. The assay was linear over the range 28-2800 ng/mL (r(2) = 0.9993) for aloe emodin and 25.6-2560 ng/mL (r(2) = 0.9991) for chrysophanol. The average percentage recoveries of three spiked plasmas were 98.8-104.8% and 97.7-103.2% for aloe emodin and chrysophanol, respectively. Their RSD of intra-day and inter-day precision at concentrations of 56, 280 and 1400 ng/mL for aloe emodin and 51.6, 258 and 1290 ng/mL for chrysophanol were less than 3.5%. This method was applied for the first time to simultaneously determinate aloe emodin and chrysophanol in rats following oral administration of traditional Chinese medicine of Da-Cheng-Qi decoction. The pharmacokinetic parameters showed that chrysophanol was better absorbed with higher concentrations in plasma than aloe emodin did. They both eliminated slowly in male rats. The assay is suitable for identifying the plasma and tissue levels of aloe emodin and chrysophanol in preclinical investigations.