LC‐ESI‐MS/MS analysis of quercetin in rat plasma after oral administration of biodegradable nanoparticles

A simple, rapid and sensitive LC‐MS/MS method was developed and validated for the determination of free quercetin in rat plasma, using fisetin as internal standard. The detection was performed by negative ion electrospray ionization under selected reaction monitoring. Chromatographic separation (isocratic elution) was carried out using acetonitrile–10 m m ammonium formate (80:20, v/v) with 0.1% v/v formic acid. The lower limit of quantification (4.928 ng/mL) provided high sensitivity for the detection of quercetin in rat plasma. The linearity range was from 5 to 2000 ng/mL. Intra‐ and inter‐day variability (RSD) of quercetin extraction from rat plasma was <4.19 and 1.37% with accuracies of 98.77 and 99.67%. The method developed was successfully applied for estimating free quercetin in rat plasma, after oral administration of quercetin‐loaded biodegradable nanoparticles (QLN) and quercetin suspension. QLN (C_max, 1277.34 ± 216.67 ng/mL; AUC, 17,458.25 ± 3152.95 ng hr/mL) showed a 5.38‐fold increase in relative bioavailability as compared with quercetin suspension (C_max, 369.2 ± 108.07 ng/mL; AUC, 3276.92 ± 396.67 ng hr/mL). Copyright © 2015 John Wiley & Sons, Ltd.     

LC–MS/MS method for simultaneous determination of serum p‐cresyl sulfate and indoxyl sulfate in patients undergoing peritoneal dialysis

p‐Cresol sulfate (pCS) and indoxyl sulfate (IS) are protein‐bound uremic toxins that accumulate in patients with chronic kidney disease (CKD). They are closely associated with the mortality rate of CKD and morbidity of cardiovascular disease. In the present study, we established a rapid method for determination of pCS and IS by HPLC‐MS/MS in serum samples from 205 CKD patients undergoing peritoneal dialysis. In brief, serum was extracted by acetonitrile and spiked with hydrochlorothiazide. The prepared sample was eluted through HPLC column (Agilent Zorbax SB‐C₁₈, 3.5 μm, 2.1 × 100 mm) with a mobile phase of acetonitrile and 10 mm ammonium acetate solution (10:90, v/v) for subsequent detection of pCS and IS by MS/MS. The linearity ranged from 50 to 10,000 ng/mL for pCS (r > 0.99), and from 500 to 10,000 ng/mL for IS (r > 0.99). The lower limit of quantification was 50 ng/mL for pCS, and 500 ng/mL for IS. Relative standard deviation (RSD) of intra‐ and inter‐day precision was within ±15%. The results showed that pCS and IS levels were partially correlated with renal function in CKD patients, and IS was directly related to serum creatinine and estimated glomerular filtration rate.     

Liquid Chromatography‐Electrospray Ionization Mass Spectrometric Method for Determination of Gliclazide in Human Plasma

Abstract

A rapid, sensitive, and specific liquid chromatography‐electrospray ionization mass spectrometric (LC‐ESI‐MS) method has been developed for quantification of gliclazide in human plasma. The analyte and tolbutamide (internal standard, I.S.) were extracted from plasma samples with n‐hexane–dichloromethane (1:1, v/v) and analyzed on a C₁₈ column. The chromatographic separation was achieved within 4.0 min by using methanol–0.5% formic acid (80:20, v/v) as mobile phase and the flow rate was 1.0 mL/min. Ion signals m/z 324.0 and 271.0 for gliclazide and internal standard were measured in the positive mode, respectively. The method was linear within the range of 2.5–2000 ng/mL. The lower limit of quantification (LLOQ) was 2.5 ng/mL. The intra‐ and inter‐day precisions were lower than 2.8% in terms of relative standard deviation (RSD). The inter‐day relative error (RE) as determined from quality control samples (QCs) ranged from ?1.93% to 1.85%. This validated method was successfully applied for the evaluation of pharmacokinetic profiles of gliclazide modified‐release tablets in 20 healthy volunteers.     

Simultaneous determination of serum propafenone and its metabolites using high‐performance liquid chromatography

Propafenone, a class Ic antiarrhythmic agent, is metabolized to 5‐hydroxypropafeone (5‐OHP) and N‐depropylpropafenone (NDPP). Simultaneous determination of serum propafenone and its metabolites was performed using HPLC equipped with a conventional octadecylsilyl silica column and ultraviolet detector. The wavelength was set at 250 nm. Propafenone and its metabolites in the serum were extracted using diethyl ether. The mobile phase solution, comprising 1‐pentanesulfonic acid sodium salt (0.1 m ), acetonitrile and acetic acid (280:185:2.5, v/v/v), was pumped at a flow rate of 1 mL/min. The recoveries of propafenone, 5‐OHP and NDPP were greater than 85, 82 and 60%, respectively, with the coefficients of variation (CVs) less than 5.4, 1.9 and 2.9%, respectively. The calibration curves were linear for a concentration range of 12.5–1500 ng/mL for propafenone and 2–500 ng/mL for 5‐OHP and NDPP (r > 0.999). CVs in the intraday assays were 1.0–3.8% for propafenone, 0.6–2.0% for 5‐OHP and 0.6–1.7% for NDPP. CVs in interday assays were 1.3–7.7% for propafenone, 1.1–6.5% for 5‐OHP and 5.4–8.0% for NDPP. The present HPLC method can be used to assess the disposition of propafenone and its metabolites for pharmacokinetic studies and therapeutic drug monitoring of propafenone.     

Facile LC‐UV methods for simultaneous monitoring of ciprofloxacin and rosuvastatin in API,formulations and human serum

An efficient, selective and cost‐effective liquid chromatographic assay was developed and validated for the simultaneous quantification of ciprofloxacin and rosuvastatin in Active Pharmaceutical Ingredients (API), pharmaceutical formulations and in human serum. The chromatographic system consisted of mobile phase methanol–water, 90:10 v/v at pH 3.0 adjusted with o‐phosphoric acid, pumped at 1.0 mL/min through a prepacked Purospher Star C18 (5 µm, 25 × 0.46 cm) column and effluent was monitored at the isosbestic point (255 nm) as well as at the λ_max of individual drugs (243 and 271 nm). The method was validated over a linear concentration range of 0.25–15 µg/mL for ciprofloxacin and 0.33–20 µg/mL for rosuvastatin (r² ≥ 0.999). The ranges of reliable response (limits of detection and quantitation) for ciprofloxacin were 3–15 and 9–45 ng/mL and 17–29 and 52–88 ng/mL, respectively, for rosuvastatin in all API, pharmaceutical formulations and human serum. Analytical recovery from human serum was >98% and relative standard deviation (RSD) was <2. The accuracies were 97.13–102.55 and 97.41–101.31% and precisions in RSD were 0.04–1.90 and 0.02–1.23% for ciprofloxacin and rosuvastatin, respectively. No matrix interferences, ion suppression/enhancement and carry‐over were detected. The total assay run time was less than 5 min. In another study, for optimum performance the detector was programmed for multiwavelength scanning at the absorption maxima of each component. Consequently, the linearity range was improved and limit of detection and quantitation values were down to 1–4 and 4–12 ng/mL for ciprofloxacin and 3–5 and 9–15 ng/mL for rosuvastatin, respectively. The validation parameters fitted ICH guidelines through the isosbestic and individual λ_max approach. The small sample volume and simplicity of preparation make this method suitable for use in human serum samples, pharmaceutical formulations, quality control, drug–drug interaction studies, clinical laboratories, drug research centers and forensic medical centers. Copyright © 2014 John Wiley & Sons, Ltd.     

Comparative Study of the Determination of Parabens in Shampoos by Liquid Chromatography with Amperometric and Coulometric Detection

The simultaneous determination of four para‐hydroxybenzoic acid esters (parabens) in shampoos was studied by liquid chromatography (LC) with amperometric (LC‐AD) and coulometric (LC‐CD) detection. The parabens were separated on an ODS C18 reversed column by isocratic elution with a mobile phase based on methanol‐0.1 M acetic acid (60 : 40%, v/v) with 0.02 M NaClO₄ at a flow rate of 0.8 mL min^?1. The limit of detection (S/N>3) for the analytes was in the 15–25 pg (injected mass) range at an applied potential of 1.20 V vs. Ag/AgCl using the LC‐AD and in the 2–3 pg range at a potential of 0.790 V vs. Pd using the LC‐CD. The peak ratio of the internal standard peak (IS: 4‐hydroxybenzoic acid sec‐butyl ester) versus the analyte peak was found to be related to the amount injected from 0.1 ng to 100ng (r=0.996–0.999) with the LC‐AD and from 0.050 ng to 100 ng range (r=0.999–1.000) with the LC‐CD. The relative standard deviation (RSD, n=10) was comprised between 1.8 to 3.5% by LC‐AD ( 5 ng injected) and between 2.0 to 2.4% by LC‐CD (0.5 ng injected). The determination of four most used parabens in ten different shampoos was successfully realized.     

Simultaneous quantification of sildenafil and N‐desmethyl sildenafil in human plasma by UFLC coupled with ESI‐MS/MS and pharmacokinetic and bioequivalence studies in Malay population

A simple, rapid, specific and reliable UFLC coupled with ESI‐MSMS assay method to simultaneously quantify sildenafil and N‐desmethyl sildenafil, with loperamide as internal standard, was developed. Chromatographic separation was performed on a Thermo Scientific Accucore C₁₈ column with an isocratic mobile phase composed of 0.1% v/v formic acid in purified water–methanol (20:80, v/v), at a flow rate of 0.3 mL/min. Sildenafil, N‐desmethyl sildenafil and loperamide were detected with proton adducts at m/z 475.4 > 58.2, 461.3 > 85.2 and 477.0 > 266.1 in multiple reaction monitoring positive mode, respectively. Both analytes and internal standard were extracted by diethyl ether. The method was validated over a linear concentration range of 10–800 ng/mL for sildenafil and 10–600 ng/mL for N‐desmethyl sildenafil with correlation coefficient (r²) ≥0.9976 for sildenafil and (r²) ≥0.9992 for N‐desmethyl sildenafil. The method was precise, accurate and stable. The proposed method was applied to study the bioequivalence between a 100 mg dose of two pharmaceutical products: Viagra (original) and Edyfil (generic) products. AUC_0–t, C_max and T_max were 2285.79 ng h/mL, 726.10 ng/mL and 0.94 h for Viagra and 2363.25 ng h/mL, 713.91 ng/mL and 0.83 hour for Edyfil. The 90% confidence interval of these parameters of this study fall within the regulatory range of 80–125%, hence they are considered as bioequivalent. Copyright © 2014 John Wiley & Sons, Ltd.     

Highly sensitive determination of Schisandrin and Schisandrin B in plasma of rats after administration of Wurenchun (Fructus Schisandrae Chinensis Extracts) preparations by LC‐ESI‐MS/MS

A sensitive and specific method based on liquid chromatography‐tandem mass spectrometry using electrospray ionization (LC‐ESI‐MS/MS) has been developed for the determination of Schisandrin and Schisandrin B in rat plasma. A 100 μL plasma sample was extracted by methyl tert‐butyl ether after spiking the samples with nimodipine (internal standard) and performed on an XTerra®MS‐C₁₈ column (150 mm × 2.1 mm, 3.5 μm) with the mobile phase of acetonitrile–water–formic acid (80:20:0.2, v/v) at a flow rate of 0.2 mL/min in a run time of 8.5 min. The lower limit of quantification of the method was 40 ng/mL for Schisandrin and 20 ng/mL for Schisandrin B. The method showed reproducibility with intra‐day and inter‐day precision of less than 13.8% RSD, as well as accuracy, with inter‐ and intra‐assay accuracies between 93.5 and 107.2%. Finally, the LC‐ESI‐MS/MS method was successfully applied to study the pharmacokinetics of Schisandrin and Schisandrin B in rats after administration of Wurenchun commercial formulations to rats. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

Determination of sertraline in rat plasma by HPLC and fluorescence detection and its application to in vivo pharmacokinetic studies

A simple, rapid, and sensitive HPLC method based on 9H‐fluoren‐9‐ylmethyl chloroformate derivatization for the quantification of sertraline in rat plasma has been developed, requiring a plasma sample of only 0.1 mL, which was deproteinized and derivatized for 5 min in two single steps. The obtained derivative was stable at room temperature and was determined by HPLC using a fluorescence detector. The analytical column was a C(18) column and the mobile phase was acetonitrile and water (80:20, v/v). Calibration curves were linear in the range of 10–500 ng/mL. The limit of detection was approximately 3 ng/mL, and the lower limit of quantification was established at 10 ng/mL. The bias of the method was lower than 10%, and the within day as well as between day, relative standard deviations were lower than 12%. This analytical method was successfully applied to characterize sertraline pharmacokinetics in rats following intravenous (t_1/2 = 213 ± 48 min, Cl = 43.1 ± 8.7 mL/min, V_d = 11560 ± 1861 mL) and oral (C_max = 156 ± 76 ng/mL, t_max = 63.8 ± 16.3 min) administration of 2 and 5 mg, respectively.     

Determination of zidovudine using anion exchange chromatography with integrated pulsed amperometric detection

A rapid and practical method for direct detection of zidovudine in high performance anion exchange chromatography(HPAEC) has been developed with integrated pulsed amperometric detection(IPAD).Dionex AS 18(250 mm×2 mm) and AG 18 (50 mm x 2 mm) columns and 11 mmol/L NaOH solution were used for separation.Multi-step potential waveform parameters were optimized to maximize the signal-to-noise ratio(S/N).Utilizing an optimized waveform,the repeatability(intra-day) precision and intermediate(inter-day) precision are obtained with relative standard deviation(RSD) of 1.88,2.27,respectively.The limit of quantification(LOQ) and limit of detection(LOD) were found to be 9.70,3.0 ng/mL,respectively,with correlation coefficients of 0.9992 over concentration range 0.01-10μg/mL.The present method was successfully applied to the determination of zidovudine in human plasma.The recoveries of plasma sample spiked by 0.7μg/mL,2.7μg/mL obtained were 95.3-101.5%, with relative standard deviation(RSD) of 2.54%,2.21%,respectively.     

RP-HPLC method with electrochemical detection for the determination of metoclopramide in serum and its use in pharmacokinetic studies.

A rapid and sensitive reversed-phase high performance liquid chromatographic method has been developed for the determination of metoclopramide in serum. The assay was performed after single extraction with ethyl ether using methyl parahydroxybenzoate as internal standard. Chromatographic separations were performed on C(18) stationary phase with a mobile phase composed of methanol-phosphate buffer pH 3 (30:70 v/v). Analytes were detected electrochemically. The quantification limit for metoclopramide in serum was 2 ng mL(-1). Linearity of the method was confirmed in the range of 5-120 ng mL(-1) (correlation coefficient 0.9998). Within-day relative standard deviations (RSDs) ranged from 0.3 to 5.5% and between-day RSDs from 0.8 to 6.0%. The analytical method was successfully applied for the determination of pharmacokinetic parameters after ingestion of 10 mg dose of metoclopramide. Studies were performed on 18 healthy volunteers of both sexes.     

Validation of a sensitive HPLC/fluorescence method for assessment of ciprofloxacin levels in plasma and prostate microdialysate samples from rats

Chronic bacterial prostatitis treatment consists of broad‐spectrum antibiotic therapy for long periods of time. Drug penetration into the prostate makes the treatment a challenged. Ciprofloxacin is one of the most prescribed drugs for this treatment. A liquid chromatography with fluorescence detection method was developed and validated for determining ciprofloxacin concentrations in two different matrices: plasma and prostate microdialysate. Ciprofloxacin was separated on a C₁₈ column eluted with a mobile phase constituted of a mixture of 0.4% aqueous triethylamine:methanol:acetonitrile (75:15:10, v/v/v) and 0.4% aqueous triethylamine:acetonitrile (88:12, v/v) for microdialysate and plasma samples, respectively. Linearity was obtained over a concentration range of 5–1000 ng/mL (microdialysate) and 10–2000 ng/mL (plasma), with coefficients of determination ≥0.9956. Precision was determined from the analysis of six quality control samples and showed RSD values <11.1 and 7.4% for intra and inter‐assay precision, respectively. The accuracy ranged from 85.6 to 114.3%. The method was applied to a preliminary pharmacokinetic study to investigate ciprofloxacin concentrations in prostate, sampled by microdialysis, and plasma after a 7 mg/kg intravenous dose to Wistar rats. The method showed high sensitivity using only protein precipitation as plasma sample clean‐up and was successfully applied to investigate ciprofloxacin prostate penetration. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of the stress biomarker corticosterone in serum of tumor‐bearing mice by surrogate‐based liquid chromatography–tandem mass spectrometry

A simple, robust and specific liquid chromatography tandem mass spectrometry (LC‐MS/MS) method was developed and validated to determine the concentration of corticosterone (Cort) which is usually regarded as a stress biomarker in mouse serum. Since Cort is an endogenous hormone, a ‘surrogate analyte’ strategy was adopted using the stable isotope‐deuterated corticosterone as a surrogate of the authentic analyte to generate the calibration curve. With telmisartan as the internal standard, the analytes were extracted with methanol, ethanol and acetone (1:1:1, v/v/v) and separated on a XTerra C₁₈ (2.1 × 50 mm, 3.5 µm) column using a mobile phase consisting of 0.2% formic acid in water–methanol (30:70, v/v). Detection was performed in multiple reaction monitoring mode with an electrospray ionization source operated in positive ion mode. The standard curves were linear (r² > 0.999) over the dynamic range of 8.60–430 ng/mL, with a lower limit of quantification of 8.60 ng/mL. The intra‐ and inter‐assay precisions were less than 15.0% of the relative standard deviation. This method was further used for analysis of serum samples from C57B/L tumor‐bearing mice before and after the treatment of fluoxetine. Validation of the assay and its application to the analysis demonstrated that the method was applicable to determine meaningful changes in Cort concentrations in serum samples of the tumor‐bearing mice for the stress status evaluation. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of metoprolol in human plasma and urine by high‐performance liquid chromatography with fluorescence detection

A simple, specific and sensitive HPLC method has been developed for the determination of metoprolol in human plasma and urine. Separation of metoprolol and atenolol (internal standard) was achieved on an Ace C₁₈ column (5 μm, 250 mm×4.6 mm id) using fluorescence detection with λ_ex=276 nm and λ_em=296 nm. The mobile phase consists of methanol–water (50:50, v/v) containing 0.1% TFA. The analysis was performed in less than 10 min with a flow rate of 1 mL/min. The assay was linear over the concentration range of 3 – 200 and 5 – 300 ng/mL for plasma and urine, respectively. The LOD were 1.0 and 1.5 ng/mL for plasma and urine, respectively. The LOQ were 3.0 and 5.0 ng/mL for plasma and urine, respectively. The extraction recoveries were found to be 95.6 ± 1.53 and 96.4 ± 1.75% for plasma and urine, respectively. Also, the method was successfully applied to three patients with hypertension who had been given an oral tablet of 100 mg metoprolol.     

Determination of tubuloside B by LC‐MS/MS and its application to a pharmacokinetic study in rats

Tubuloside B, a novel neuroprotective phenylethanoid, is a major active constituent of Cistanche tubulosa and Cistanche deserticola. A specific and sensitive liquid chromatography tandem mass spectrometry (LC‐MS/MS) method has been developed and validated for the quantification of tubuloside B in rat plasma. Sample preparation was conducted through a protein‐precipitation extraction with methanol using tubuloside A as internal standard (IS). Chromatographic separation was achieved using a Capcell Pak C₁₈ column (2.0 × 50 mm, 5 μm) with a mobile phase of methanol–10 mm ammonium acetate buffer (70:30, v/v) in an isocratic elution. Mass spectrometry analysis was performed in negative ionization mode with selected reaction monitoring transitions at m/z 665.1 → 160.9 for tubuloside B, and m/z 827.1 → 160.9 for IS. Calibration curves were linear over the range of 1.64–1640 ng/mL for plasma samples samples (R² > 0.990). The lower limit of quantification (LLOQ) was 1.64 ng/mL. The intra‐ and inter‐day accuracy was between 92.3 and 113.0% with the RSD <9.23% at all LLOQ and quality control levels. Finally, this method was successfully applied in the pharmacokinetics study of tubuloside B after intravenous administration.     

Determination of ibudilast in human serum by high‐performance liquid chromatography for pharmacokinetic study

A simple, accurate, precise and cost effective reversed‐phase HPLC method was developed to determine the concentration of ibudilast in human serum. Ibudilast and an internal standard, butyl 4‐hydroxybenzoate, were extracted by liquid–liquid extraction with methyl tert‐butyl ether. HPLC analysis was carried out under the following conditions: a Luna C₁₈(2) 5 μm column, a mobile phase of acetonitrile–0.02% phosphoric acid (50 : 50, v/v, adjusted to pH 6.0 with triethylamine) and a UV detector at 319 nm. The chromatograms showed good resolution and sensitivity as well as no interference from the human serum. The calibration curves were linear over the concentration range, 1–100 ng/mL, for serum with correlation coefficients >0.999. The intra‐ and inter‐day assay precision as well as the accuracy fulfilled the international requirements. The mean absolute recovery for human serum was 101.7 ± 6.1%. The lower limit of quantitation in human serum was 1 ng/mL, which is sensitive enough for pharmacokinetic studies. Stability studies revealed that ibudilast in human serum was stable during storage as well as during the assay procedure. This method was applied successfully to an examination of the pharmacokinetics of ibudilast in human subjects following a single oral dose of an ibudilast (10 mg) capsule. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and validation of a UPLC–MS method for the determination of galantamine in guinea pig plasma and its application to a pre‐clinical bioavailability study of novel galantamine formulations

To evaluate the bioavailability and pharmacokinetic profiles of two novel galantamine formulations as medical countermeasure products, an ultra‐performance liquid chromatography–single quadrupole mass spectrometry (UPLC–MS) method was developed and validated for quantifying galantamine in guinea pig plasma using solid‐phase extraction with a mixed mode strong cation exchange reversed‐phase cartridge. Chromatographic separation was achieved on a Waters Acquity UPLC BEH C₁₈ column maintained at 40°C. The mobile phases were solution A, acetonitrile–water, 5:95 (v/v) and solution B, acetonitrile–water 90:10 (v/v), both containing 2 mM ammonium formate and 0.2% formic acid. The mobile phase was delivered utilizing a 3 min gradient program start with 95%A–5%B at a flow rate of 0.6 mL/min. The analyte and internal standard, galantamine‐d3, were detected by selected ion monitoring mode on a Waters 3100 single quadrupole mass spectrometer with positive electrospray ionization. The method was validated according to the US Food and Drug Administration bioanalytical guidance. The method was selective and was linear over the analytical range of 2–2000 ng/mL. Accuracy and precision were acceptable with intra‐ and inter‐day accuracies between 96.8 and 101% and precisions (RSD) <4.88%. The method was successfully implemented to measure galantamine plasma levels in a series of pre‐clinical bioavailability studies for the evaluation of novel galantamine formulations.     

Development and validation of a rapid and efficient method for simultaneous determination of methylxanthines and their metabolites in urine using monolithic HPLC columns

A new rapid, sensitive and validated HPLC method has been developed for the determination of methylxanthines and their metabolites in asthmatic patients. The method was initiated by using spiked urine samples on a silica monolithic column as a novel packing material. The mobile phase consisted of 10 mM potassium dihydrogen phosphate buffer/methanol (87.5:12.5 v/v), at a flow rate 1 mL/min. Detection was set at 274 nm. The LOQ for all the compounds ranged from 14 to 41 ng/mL. Excellent linearity was achieved over the studied range of concentration with correlation coefficients 0.9991–0.9998 (n = 6). The developed method was validated by precision and accuracy with RSD <2.55%. On extraction of the drugs and metabolites from the urine samples high recoveries were achieved ranging from 82.06 to 98.34% w/w on RP18 cartridges and methanol/chloroform (20:80 v/v) as the extraction solvent. This method has advantages over other methods using conventional C18 packings.     

A Unique Solid Phase Extraction Column for Isolation of 11-Nor-Δ-9-Tetrahydrocannabinol-9-Carboxylic Acid in Human Urine

Abstract

A sensitive, specific, qualitative, and quantitative extraction procedure followed by an hplc assay of 11-nor-Δ-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) from human urine is developed. Using a new, “mixed mode”, bonded silica gel solid phase extraction (SPE) column, the analyte was selectively isolated from the urine component. Following extraction, the presence of THC-COOH was confirmed and quantitated by a UV detector on a Varian 15cm C18 column using 35:65 v/v 50 mM phosphoric acid:acetonitrile at a flow rate of 1.5 mL/min. The limit of detection was 10 ng/mL at a signal to noise ratio of 2.5. The method showed linearity in the 10–300 ng/mL range (r=0.999) with good precision (RSD 1.4%) and accuracy (87% absolute recovery).