Synthesis and study of a molecularly imprinted polymer for the specific extraction of indole alkaloids from Catharanthus roseus extracts

Two molecularly imprinted polymers (MIP) for catharanthine and vindoline have been synthesized in order to specifically extract these natural indole alkaloids from Catharanthus roseus by solid-phase extraction (SPE). Each MIP was prepared by thermal polymerisation using catharanthine (or vindoline) as template, methacrylic acid (or itaconic acid) as functional monomer, ethylene glycol dimethacrylate (EDMA) as cross-linking agent and acetonitrile (or acetone) as porogenic solvent.For catharanthine-MIP, a SPE protocol (ACN–AcOH 99/1 washing and MeOH–AcOH 90/10 elution) allows a good MIP/NIP selectivity (imprinting factor 12.6). The specificity of catharanthine-MIP versus related bisindole alkaloids was assessed by cross-reactivity study. The catharanthine-MIP specifically retained catharanthine and its N-oxide analogue but displayed a weak cross-reactivity for other Vinca alkaloids (vinorelbine, vincristine, vinblastine, vindoline, vinflunine). It appears that the catharanthine-like unit of these molecules are hardly trapped in catharanthine cavities located in the MIP, probably due to the sterical hindrance of the vindoline moiety. Finally, the MIP-SPE applied to C. roseus extract enabled quantitative recovery of catharanthine (101%) and the total removal of vindoline. Its capacity was determined and was equal to 2.43 μmol g⁻¹.Vindoline is a weaker base than catharanthine, so the vindoline-MIP was achieved with a strong acidic monomer (itaconic acid) to increase vindoline–monomer interactions and a modified washing solvent (ACN–HCOOH 99/1) to reduce non-specific interactions. The influence of the amount of HCOOH (protic modifier) percolated during the washing step upon the elution yield and the imprinting factor for vindoline was investigated. This preliminary optimisation of the washing step, and in particular the number of moles of acid percolated, seems useful to emphasize the use of MIP in conditions of high selectivity or high yield. A compromise was obtained with an imprinting factor equal to 7.6 and an elution recovery of 33%. However MIP-vindoline failed to achieve a specific extraction of vindoline since catharanthine was also extracted probably because of strong non-specific interactions occurring between catharanthine and the sorbent.     

Synthesis of molecularly imprinted polypyrrole as an adsorbent for solid‐phase extraction of warfarin from human plasma and urine

The aim of this work was to develop a method for the clean‐up and preconcentration of warfarin from biological sample employing a new molecularly imprinted polymer (MIP) as a selective adsorbent for solid‐phase extraction (SPE). This MIP was synthesized using warfarin as a template, pyrrole as a functional monomer and vinyl triethoxysilane as a cross‐linker. The molar ratio of 1:4:20 (template–functional monomer–cross‐linker) showed the best results. Nonimprinted polymers (NIPs) were prepared and treated with the same method, but in the absence of warfarin. The prepared polymer was characterized by Fourier transmission infrared spectrometry and scanning electron microscopy. An adsorption process (SPE) for the removal of warfarin using the fabricated MIPs and NIPs was evaluated under various conditions. Effective parameters on warfarin extraction, for example, type and volume of elution solvent, pH of sample solution, breakthrough volume and maximum loading capacity, were studied. The limits of detection were in the range of 0.0035–0.0050 µg mL^?1. Linearity of the method was determined in the range of 0.0165–10.0000 µg mL^?1 for plasma and 0.0115–10.0000 µg mL^?1 for urine with coefficients of determination (R²) ranging from 0.9975 to 0.9985. The recoveries for plasma and urine samples were >95%. Copyright © 2015 John Wiley & Sons, Ltd.     

Selective extraction of clonazepam from human plasma and urine samples by molecularly imprinted polymeric beads

A molecularly imprinted polymer (MIP) based on free‐radical polymerization was prepared with 1‐(N,N‐biscarboxymethyl)amino‐3‐allylglycerol and N,N‐dimethylacrylamide as functional monomers, N,N‐methylene diacrylamide as the cross‐linker, copper ion‐clonazepam as the template and 2,2‐azobis(2‐methylbutyronitrile) as the initiator. The imprinted polymer was characterized by Fourier transform infrared spectroscopy, elemental analysis, thermogravimetric analysis, and SEM. The MIP of agglomerated microparticles with multipores was used for SPE. The imprinted polymer sorbent was selective for clonazepam. The optimum pH and sorption capacity were 5 and 0.18 mg/g at 20°C, respectively. The profile of the drug uptake by the sorbent reflects good accessibility of the active sites in the imprinted polymer sorbent. The MIP‐SPE was the most feasible technique for the extraction of clonazepam with a high recovery from human plasma and urine samples.     

A (−)‐norephedrine‐based molecularly imprinted polymer for the solid‐phase extraction of psychoactive phenylpropylamino alkaloids from Khat (Catha edulis Vahl. Endl.) chewing leaves

A molecularly imprinted polymer (MIP) was prepared using (?)‐norephedrine as the template, methacrylic acid as the functional monomer, ethylene glycol dimethacrylate as the cross‐linker and chloroform as the porogen. The MIP was used as a selective sorbent in the molecularly imprinted solid‐phase extraction (MIP‐SPE) of the psychoactive phenylpropylamino alkaloids, norephedrine and its analogs, cathinone and cathine, from Khat (Catha edulis Vahl. Endl.) leaf extracts prior to HPLC‐DAD analysis. The MIP was able to selectively extract the alkaloids from the aqueous extracts of Khat. Loading, washing and elution of the alkaloids bound to the MIP were evaluated under different conditions. The clean baseline of the Khat extract obtained after MIP‐SPE confirmed that a selective and efficient sample clean‐up was achieved. Good recoveries (90.0–107%) and precision (RSDs 2.3–3.2%) were obtained in the validation of the MIP‐SPE‐HPLC procedure. The content of the three alkaloids in Khat samples determined after treatment with MIP‐SPE and a commercial Isolute C₁₈ (EC) SPE cartridge were in good agreement. These findings indicate that MIP‐SPE is a reliable method that can be used for sample pre‐treatment for the determination of Khat alkaloids in plant extracts or similar matrices and could be applicable in pharmaceutical, forensic and biomedical laboratories. Copyright © 2015 John Wiley & Sons, Ltd.     

Quantification and application of a liquid chromatography–tandem mass spectrometric method for the determination of WKYMVm peptide in rat using solid‐phase extraction

A liquid chromatographic–electrospray ionization–time‐of‐flight/mass spectrometric (LC‐ESI‐TOF/MS) method was developed and applied for the determination of WKYMVm peptide in rat plasma to support preclinical pharmacokinetics studies. The method consisted of micro‐elution solid‐phase extraction (SPE) for sample preparation and LC‐ESI‐TOF/MS in the positive ion mode for analysis. Phenanthroline (10 mg/mL) was added to rat blood immediately for plasma preparation followed by addition of trace amount of 2 m hydrogen chloride to plasma before SPE for stability of WKYMVm peptide. Then sample preparation using micro‐elution SPE was performed with verapamil as an internal standard. A quadratic regression (weighted 1/concentration²), with the equation y = ax² + bx + c was used to fit calibration curves over the concentration range of 3.02–2200 ng/mL for WKYMVm peptide. The quantification run met the acceptance criteria of ±25% accuracy and precision values. For quality control samples at 15, 165 and 1820 ng/mL from the quantification experiment, the within‐run and the between‐run accuracy ranged from 92.5 to 123.4% with precision values ≤15.1% for WKYMVm peptide from the nominal values. This novel LC‐ESI‐TOF/MS method was successfully applied to evaluate the pharmacokinetics of WKYMVm peptide in rat plasma.     

A rapid and sensitive LC-MS/MS method for quantification of donepezil and its active metabolite, 6-o-desmethyl donepezil in human plasma and its pharmacokinetic application

A rapid and sensitive liquid chromatography–tandem mass spectrometric (LC‐MS/MS) assay method has been developed and fully validated for simultaneous quantification of donepezil and its active metabolite, 6‐o‐desmethyl donepezil in human plasma. Analytes and the internal standard were extracted from human plasma by liquid–liquid extraction technique using a 30:70 v/v mixture of ethyl acetate and n‐hexane. The reconstituted samples were chromatographed on a C₁₈ column by using a 70:30 v/v mixture of acetonitrile and ammonium formate (5 mm , pH 5.0) as the mobile phase at a flow rate of 0.6 mL/min. The calibration curve obtained was linear (r ≥ 0.99) over the concentration range of 0.09–24.2 ng/mL for donepezil and 0.03–8.13 ng/mL for 6‐o‐desmethyl donepezil. The results of the intra‐day and inter‐day precision and accuracy studies were well within the acceptable limits. The proposed method was successfully applied for the estimation of the drug in real time plasma samples for pharmacokinetic studies. Copyright © 2010 John Wiley & Sons, Ltd.     

A rapid MCM‐41 dispersive micro‐solid phase extraction coupled with LC/MS/MS for quantification of ketoconazole and voriconazole in biological fluids

A rapid dispersive micro‐solid phase extraction (D‐μ‐SPE) combined with LC/MS/MS method was developed and validated for the determination of ketoconazole and voriconazole in human urine and plasma samples. Synthesized mesoporous silica MCM‐41 was used as sorbent in d ‐μ‐SPE of the azole compounds from biological fluids. Important D‐μ‐SPE parameters, namely type desorption solvent, extraction time, sample pH, salt addition, desorption time, amount of sorbent and sample volume were optimized. Liquid chromatographic separations were carried out on a Zorbax SB‐C₁₈ column (2.1 × 100 mm, 3.5 μm), using a mobile phase of acetonitrile–0.05% formic acid in 5 mm ammonium acetate buffer (70:30, v /v). A triple quadrupole mass spectrometer with positive ionization mode was used for the determination of target analytes. Under the optimized conditions, the calibration curves showed good linearity in the range of 0.1–10,000 μg/L with satisfactory limit of detection (≤0.06 μg/L) and limit of quantitation (≤0.3 μg/L). The proposed method also showed acceptable intra‐ and inter‐day precisions for ketoconazole and voriconazole from urine and human plasma with RSD ≤16.5% and good relative recoveries in the range 84.3–114.8%. The MCM‐41‐D‐μ‐SPE method proved to be rapid and simple and requires a small volume of organic solvent (200 μL); thus it is advantageous for routine drug analysis.     

Preparative isolation of alkaloids from Corydalis bungeana Turcz. by high-speed counter-current chromatography using stepwise elution

High‐speed counter‐current chromatography (HSCCC) was successfully applied for the preparative separation and purification of alkaloids from Corydalis bungeana Turcz. (Kudiding in Chinese) for the first time. After the measurement of partition coefficient of seven target alkaloids in the nine two‐phase solvent systems composed of CHCl₃–MeOH–(0.1 M; 0.2 M; 0.3 M) HCl (4:1.5:2; 4:2:2; 4:3:2, v/v), CHCl₃–MeOH–0.2 M HCl (4:2:2, v/v) and CHCl₃–MeOH–0.3 M HCl (4:3:2, v/v) were finally selected for the HSCCC separation using the first upper phase as the stationary phase and the stepwise elution of the two lower mobile phases. Consequently, sanguinarine (10 mg), corynoline (25 mg), protopine (20 mg), corynoloxine (18 mg), and 12‐hydroxycorynoline (8 mg) were obtained from 200 mg of crude alkaloid extracts with purities of 94–99% as determined by HPLC. Their chemical structures were characterized on the basis of ¹H‐NMR, ¹³C‐NMR, and LC‐ESI‐Q‐TOF‐MS/MS analyses.     

A validated LC-MS/MS method for the determination of vinflunine in plasma and its application to pharmacokinetic studies

A rapid, simple and sensitive LC‐MS/MS method for the quantification of vinflunine in plasma was developed and validated. The analysis involved a simple liquid–liquid extraction. After making alkaline with NaOH, plasma was extracted with methyl tert‐butyl ether and the organic extract was then evaporated and the residue was reconstituted in mobile phase. The reconstituted solution was injected into an HPLC system and was subjected to reverse‐phase HPLC on a 5 µm ODS‐3 column at a flow‐rate of 0.2 mL/min. The mobile phase consisted of ammonium acetate (0.02 mol/L, pH = 3.0) and acetonitrile (20:80). Vinflunine was detected in the single ion monitoring mode using target ions at m/z 817.4/160.1/142.3 for vinflunine and m/z 447.2/128.3/112.1 for gefitinib (internal standard). Standard curves were linear over the concentration range of 5–1000 ng/mL. The mean predicted concentrations of the quality control samples deviated by less than 2% from the corresponding nominal values; the intra‐assay and inter‐assay precisions of the assay were within 7% relative standard deviation. The extraction recovery of vinflunine was more than 80%. The validated assay was applied to a pharmacokinetic study of vinflunine in plasma following the administration of a single vinflunine injection (2 mg/kg). Copyright © 2011 John Wiley & Sons, Ltd.     

Preparation and utilization of a molecularly imprinted polymer for solid phase extraction of tramadol

In this paper, a highly selective molecularly imprinted polymer (MIP) for tramadol hydrochloride, a drug used to treat moderate to severe pain, was prepared and its use as solid-phase extraction (SPE) sorbent was demonstrated. The molecularly imprinted solid-phase extraction procedure followed by high performance liquid chromatography with ultraviolet detector (MISPE-HPLC) was developed for selective extraction and determination of tramadol in human plasma and urine. The optimal conditions for molecularly imprinted solid-phase extraction (MISPE) consisted of conditioning with 1 mL methanol and 1 mL of deionized water at neutral pH, loading of tramadol sample (50 μg L⁻¹) at pH 7.5, washing using 1 mL acetone and elution with 3 × 1 mL of 10% (v/v) acetic acid in methanol. The MIP selectivity was evaluated by checking several substances with similar molecular structures to that of tramadol. Results from the HPLC analyses showed that the calibration curve of tramadol (using MIP from human plasma and urine) is linear in the ranges of 6–100 and 3–120 μg L⁻¹ with good precisions (1.9% and 2.9% for 5.0 μg L⁻¹), respectively. The recoveries for plasma and urine samples were higher than 81%.      

A novel and sensitive UPLC–MS/MS method to determine mequitazine in rat plasma and urine: Validation and its application to pharmacokinetic studies

The aim of this study was to develop an analytical method to determine mequitazine in rat plasma and urine. Mequitazine was separated by UPLC–MS/MS equipped with a Kinetex core–shell C₁₈ column (50 × 2.1 mm, 1.7 μm) using 0.1% (v/v) aqueous formic acid and acetonitrile containing 0.1% (v/v) formic acid as a mobile phase by gradient elution at a flow rate of 0.3 mL/min. Quantitation of this analysis was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique operating in multiple reaction monitoring positive ion mode. Mass transitions were m/z 323.3 → 83.1 for mequitazine and 281.3 → 86.3 for imipramine as internal standard. Liquid–liquid extraction with ethyl acetate and protein precipitation with methanol were used for sample extraction. Chromatograms showed that the method had high resolution, sensitivity and selectivity without interference from plasma constituents. Calibration curves for mequitazine in rat plasma and urine were 0.02–200 ng/mL, showing excellent linearity with correlation coefficients (r²) >0.99. Both intra‐ and inter‐day precisions (CV%) were within 4.08% for rat plasma and urine. The accuracies were 99.58–102.03%. The developed analytical method satisfied the criteria of international guidance. It could be successfully applied to pharmacokinetic studies of mequitazine after oral and intravenous administration to rats.     

Enantioselective separation and determination of adrafinil and modafinil on Chiralcel OJ‐H column in rat serum and urine using solid‐phase extraction followed by HPLC

A simple and rapid normal‐phase HPLC method for enantiospecific separation of a psychostimulant, adrafinil (ADL), and its metabolite modafinil (MDL) in rat serum and urine was developed. The separation was accomplished on a normal‐phase polysaccharide stationary phase Chiralcel OJ‐H using n‐hexane–ethanol (62:38 v/v) as a mobile phase at a flow rate of 1.0 mL/min. Detection was carried out at 225 nm using a photo diode array (PDA) detector. The elution order of the enantiomers was determined by a polarimeter connected in series with the PDA. ADL and its metabolite were recovered from rat serum and urine by solid phase extraction using Oasis HLB cartridges and the mean recoveries were ≥80%. The enantiomers were eluted within 15 min without any interference from endogenous substances. The calibration curves were linear (r² > 0.998) in the concentration range of 1.20–500 µg/mL for ADL and MDL. The assay was specific, accurate, precise and reproducible (intra‐ and inter‐day precisions RSDs <7.2%). ADL in rat serum was stable over three freeze–thaw cycles at ambient temperature for 4 h. The method was successfully applied to pharmacokinetic studies of adrafinil after an oral administration to rats. Copyright © 2009 John Wiley & Sons, Ltd.     

The bioanalysis of vinorelbine and 4‐O‐deacetylvinorelbine in human and mouse plasma using high‐performance liquid chromatography coupled with heated electrospray ionization tandem mass–spectrometry

A sensitive, specific and efficient high‐performance liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of vinorelbine and its metabolite 4‐O‐deacetylvinorelbine in human and mouse plasma is presented. Heated electrospray ionization was applied followed by tandem mass spectrometry. A 50 µL plasma aliquot was protein precipitated with acetonitrile–methanol (1:1, v/v) containing the internal standard vinorelbine‐d3 and 20 µL volumes were injected onto the HPLC system. Separation was achieved on a 50 × 2.1 mm i.d. Xbridge C₁₈ column using isocratic elution with 1 mm ammonium acetate–ammonia buffer pH 10.5–acetonitrile–methanol (28:12:60, v/v/v) at a flow rate of 0.4 mL/min. The HPLC run time was 5 min. The assay quantifies both vinorelbine and 4‐O‐deacetylvinorelbine from 0.1 to 100 ng/mL using sample volumes of only 50 µL. Mouse plasma samples can be quantified using calibration curves prepared in human plasma. Validation results demonstrate that vinorelbine and 4‐O‐deacetylvinorelbine can be accurately and precisely quantified in human and mouse plasma with the presented method. The assay is now in use to support (pre‐)clinical pharmacologic studies with vinorelbine in humans and mice. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of Flavonoid Markers in Honey with SPE and LC using Experimental Design

Determination of flavonoid markers quercetin, hesperetin, and chrysin, found in north Iranian citrus honey samples, was carried out by solid phase extraction (SPE) and isocratic liquid chromatographic separation using central composite design. Optimum conditions for SPE were achieved using 10 mL methanol/water (13:87, v/v, pH = 7) as the washing solvent and 4 mL methanol for elution. Good clean-up and high recovery >90% were observed for all analytes. The use of water/ACN/THF/AcOH (54:36:5:5, v/v) was found to serve as the optimum mobile phase composition and allowed for the separation of analytes from endogenous compounds present in honey. SPE parameters, such as maximum loading capacity and breakthrough volume, were also determined for each analyte. Limit of detection, linear range, recovery, repeatability of retention times, and peak heights were 3.11 × 10⁻⁸–4.44 × 10⁻⁸ g g⁻¹, 0.50–50.0 μg mL⁻¹ (R ² > 0.99), 90.7–96.9%, 3.0–3.6%, and 1.0–2.6%, respectively. Precision of the overall analytical procedure, estimated by five replicate measurements for quercetin, hesperetin and chrysin in citrus honey, as well as the relative standard deviations were 4.3%, 3.8%, and 5.5%, respectively.

     

A reliable method of liquid chromatography for the quantification of acetaminophen and identification of its toxic metabolite N-acetyl-p-benzoquinoneimine for application in pediatric studies

The aim of the present study was to develop a simple, selective and reliable method to quantify acetaminophen and its toxic metabolite N‐acetyl‐p‐benzoquinoneimine (NAPQI) for pediatric studies using 100 µL plasma samples, by reverse‐phase HPLC and UV detection. The assay was performed using a C₁₈ column and an isocratic elution with water–methanol–formic acid (70:30:0.15; v/v/v) as mobile phase. Linearity of the method was assayed in the range of 1–30 µg/mL for acetaminophen and 10–200 µg/mL for NAPQI, with a correlation coefficient r = 0.999 for both compounds, and inter‐ and intra‐day coefficients of variation of less than 13%. Several commonly co‐administered drugs were analyzed for selectivity and no interference with the determinations was observed. The detection and quantification limits for acetaminophen and NAPQI were 0.1 and 1 µg/mL, and 0.1 and 10 µg/mL respectively. The present method can be used to monitor acetaminophen levels using 100 µL plasma samples, which may be helpful when very small samples need to be analyzed, as in pharmacokinetics determination or drug monitoring in plasma in children. This assay is also able to detect the NAPQI for drug monitoring in patients diagnosed with acetaminophen intoxication. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of terbinafine in human plasma using UPLC–MS/MS: Application to a bioequivalence study in healthy subjects

A high‐throughput and sensitive ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method has been developed for the determination of terbinafine in human plasma. The method employed liquid–liquid extraction of terbinafine and terbinafine‐d7 (used as internal standard) from 100 μL human plasma with ethyl acetate–n‐hexane (80:20, v/v) solvent mixture. Chromatography was performed on a BEH C₁₈ (50 × 2.1 mm, 1.7 μm) column using acetonitrile–8.0 mm ammonium formate, pH 3.5 (85:15, v/v) under isocratic elution. For quantitative analysis, MS/MS ion transitions were monitored at m/z 292.2/141.1 and m/z 299.1/148.2 for terbinafine and terbinafine‐d7, respectively, using electrospray ionization in the positive mode. The method was validated according to regulatory guidance for selectivity, sensitivity, linearity, recovery, matrix effect, stability, dilution reliability and ruggedness with acceptable accuracy and precision. The method shows good linearity over the tested concentration range from 1.00 to 2000 ng/mL (r² ≥ 0.9984). The intra‐batch and inter‐batch precision (CV) was 1.8–3.2 and 2.1–4.5%, respectively. The method was successfully applied to a bioequivalence study with 250 mg terbinafine in 32 healthy subjects. The major advantage of this method includes higher sensitivity, small plasma volume for processing and a short analysis time.     

Development and validation of an HPLC method for the determination of seven penicillin antibiotics in veterinary drugs and bovine blood plasma

Herein a quantitative method for the determination of seven penicillins in bovine plasma and veterinary drugs has been developed. Amoxicillin (AMO), ampicillin (AMP), penicillin G (PENG), penicillin V (PENV), oxacillin (OXA), cloxacillin (CLO) and dicloxacillin (DICLO) were separated on a Perfectsil ODS‐2 (250×4 mm, 5 μm) column, using gradient elution, with a mobile phase of 0.1% v/v TFA and ACN–methanol (90:10 v/v). PDA detection was used at 240 nm. Penicillins were isolated from bovine plasma by SPE on Lichrolut RP‐18 cartridges with mean recoveries from 85.7 to 113.5%. Colchicine (3 ng/μL) was used as an internal standard. The developed method was validated in terms of selectivity, linearity, accuracy, precision, stability and sensitivity. Repeatability (n = 5) and between‐day precision (n = 5) revealed RSD < 12%. The detection limits in the bovine plasma were estimated as 18 ng for AMO and AMP, 25 for PENG, PENV and OXA, 3 ng for CLO and 12 ng for DICLO. Spiked plasma samples were stable for 1 wk, except for AMP and CLO, which were stable for 3 wk and OXA for 4 wk. AMO, PENG and PENV were stable for two freeze–thaw cycles, OXA, CLO and DICLO for four, while AMP only for one.     

Rapid and sensitive liquid chromatography with tandem mass spectrometry method for the simultaneous quantification of yonkenafil and its major metabolites in rat plasma

Yonkenafil is a promising drug for treatment of male erectile dysfunction. Previous studies showed that the piperazine‐N,N’‐deethylation metabolite, piperazine‐N‐deethylation metabolite, and piperazine‐N‐deethylation‐N,N’‐deethylation metabolite were the major metabolites of yonkenafil after extensive metabolism. We developed a sensitive and selective method for the simultaneous quantification of yonkenafil and its major metabolites using high‐throughput liquid chromatography with tandem mass spectrometry. Analytes and internal standard were extracted from a small quantity of plasma (50 μL) using liquid–liquid extraction with diethyl ether/dichloromethane (60:40, v/v), and the baseline separation was achieved on Zorbax SB‐C₁₈ column using ammonia/water/methanol (0.2:20:80, v/v/v) as the mobile phase. The assay was performed with an electrospray positive ionization mass spectrometry through the multiple‐reaction monitoring mode within 2 min. Calibration curve of the method was linear within the range of 1.00–1000 ng/mL for all the analytes with the intra‐ and interday precisions of 4.0–5.2 and 4.0–5.3% for yonkenafil, 3.1–4.9 and 3.1–5.2% for the piperazine‐N,N’‐deethylation metabolite, 4.8–6.8 and 4.8–7.3% for the piperazine‐N‐deethylation metabolite, and 2.9–6.1 and 5.4–6.3% for the piperazine‐N‐deethylation‐N,N’‐deethylation metabolite, respectively. The recoveries were above 90% with low matrix effects. The validated assay was successfully applied to support a preclinical pharmacokinetic study in six rats using a single oral dose of yonkenafil (8 mg/kg).     

Toxicological screening of human plasma by on‐line SPE‐HPLC‐DAD: identification and quantification of acidic and neutral drugs

A multi‐analyte screening method for the quantification of 50 acidic/neutral drugs in human plasma based on on‐line solid‐phase extraction (SPE)–HPLC with photodiode array detection (DAD) was developed, validated and applied for clinical investigation. Acetone and methanol for protein precipitation, three different SPE materials (two electro‐neutral, one strong anion‐exchange, one weak cation‐exchange) for on‐line extraction, five HPLC‐columns [one C₁₈ (GeminiNX), two phenyl‐hexyl (Gemini C₆‐Phenyl, Kinetex Phenyl‐Hexyl) and two pentafluorophenyl (LunaPFP(2), KinetexPFP)] for analytical separation were tested. For sample pre‐treatment, acetone in the ratio 1:2 (plasma:acetone) showed a better baseline and fewer matrix peaks in the chromatogram than methanol. Only the strong anion‐exchanger SPE cartridge (StrataX‐A, pH 6) allowed the extraction of salicylic acid. Analytical separation was carried out on a Gemini C₆‐Phenyl column (150 × 4.6 mm, 3 µm) using gradient elution with acetonitrile–water 90:10 (v/v) and phosphate buffer (pH 2.3). Linear calibration curves with correlation coefficients r ≥ 0.9950/0.9910 were obtained for 46/four analytes. Additionally, this method allows the quantification of 23 analytes for therapeutic drug monitoring. Limits of quantitation ranged from 0.1 (amobarbital) to 23 mg/L (salicylic acid). Inter‐/intra‐day precisions of quality control samples (low/high) were better than 13% and accuracy (bias) ranged from ?14 to 10%. A computer‐assisted database was created for automated detection of 223 analytes of toxicological interests. Four cases of multi‐drug intoxications are presented. Copyright © 2015 John Wiley & Sons, Ltd.     

Quantitation of unbound sunitinib and its metabolite N-desethyl sunitinib (SU12662) in human plasma by equilibrium dialysis and liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study

A rapid, selective, and sensitive liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous determination of unbound sunitinib and its active metabolite N‐desethyl sunitinib in plasma. Plasma and post‐dialysis buffer samples were extracted using a liquid–liquid extraction procedure with acetonitrile–n‐butylchloride (1:4, v/v). Chromatographic separation was achieved on a Waters X‐Terra® MS RP₁₈ column with a mobile phase consisting of acetonitrile and water (60:40, v/v) containing formic acid (0.1%, v/v) using an isocratic run, at a flow‐rate of 0.2 mL/min. Analytes were detected by electrospray tandem mass spectrometry in the selective reaction monitoring mode. Linear calibration curves were generated over the ranges 0.1–100 and 0.02–5 ng/mL for sunitinib and 0.2–200 and 0.04–10 ng/mL for N‐desethyl sunitinib in plasma and in phosphate‐buffered solution, respectively. The values for both within‐day and between‐day precision and accuracy were well within the generally accepted criteria for analytical methods. The analytical range was sufficient to determine the unbound and total concentrations of both analytes. The method was applied for measurement unbound concentrations in addition to total concentrations of sunitinib and its metabolite in plasma of a cancer patient receiving 50 mg daily dose. Copyright © 2012 John Wiley & Sons, Ltd.