Analysis of methylphenidate and its metabolite ritalinic acid in monkey plasma by liquid chromatography/electrospray ionization mass spectrometry

Methylphenidate (MP, Ritalin) is a psychotropic drug widely prescribed to children for treating the symptoms of attention deficit disorder with and without hyperactivity. Because little information exists about the effects of chronic MP administration on cognitive function in children, measures of behavior changes in non-human primates are important surrogates. An essential component of such studies is the determination of MP plasma levels under chronic and acute dosing conditions. An analytical method was developed that provided sufficient sensitivity to measure low levels of the active parent drug (lower limit of quantitation = 0.25 ng/mL) and the inactive metabolite, ritalinic acid (RA), in monkey plasma as well as the ability to conveniently analyze large numbers of samples. The method uses a polymeric reversed-phase sorbent for solid phase extraction, an efficient reverse-phase high performance liquid chromatography (HPLC) separation, deuterated internal standards for isotope dilution quantification of MP and RA, and detection by sensitive electrospray ionization mass spectrometry (ES-MS) with a single quadrupole instrument. The method responses are linear over the range of plasma concentrations of MP and RA observed in monkeys, gives respective analyte recoveries of 75 and 60% with reasonable precision and accuracy, and demonstrates robust MS performance for rapid determination of MP/RA plasma levels. The average peak MP concentration (ca. 16 ng/mL) and half-lives for MP and RA elimination in monkeys (1.79 and 2.31 h, respectively) were not significantly different under acute vs. chronic dosing conditions and were comparable to values previously reported from human studies.     

Determination of 6-mercaptopurine and azathioprine in plasma by high-performance liquid chromatography.

Using 1-ml plasma samples, levels of 6-mercaptopurine (6MP) as low as 5 ng/ml and azathioprine (AZA) as low as 40 ng/ml can be detected using a high-performance liquid chromatography reversed-phase column procedure following extraction. Both compounds were stable in frozen plasma for seven weeks. AZA stability in blood was temperature dependent; the half-lives of AZA breakdown to 6MP at 37 degrees were 28 and 46 min in blood drawn from two rhesus monkeys. Plasma levels of 6MP were measured in a rhesus monkey following 6MP (1.47 mg/kg) and AZA (3 mg/kg) intravenous administration. 6MP levels were also measured in three renal transplant patients on daily 50- and 100-mg AZA doses. Peak levels (45-75 ng/ml) were reached within an hour and 6MP levels were detected for up to 7 h.     

Direct determination of codeine-6-glucuronide in plasma and urine using solid-phase extraction and high-performance liquid chromatography with fluorescence detection

A sensitive and selective method was developed for the direct determination of codeine-6-glucuronide in plasma and urine using high-performance liquid chromatography (HPLC) with fluorescence detection. Codeine-6-glucuronide was synthesised and its purity estimated using acid and enzyme hydrolysis. The hydrolysis of codeine-6-glucuronide by beta-glucuronidase was incomplete and urine reduced the extent of hydrolysis. Codeine-6-glucuronide was recovered from plasma using a solid-phase extraction column and separated on a reversed-phase C18 HPLC column. The assay showed good reproducibility and accuracy (within 10%), and standard curves were linear between 32 and 1600 ng/ml in plasma and between 0.32 and 160 micrograms/ml in urine. The assay has been applied to the study of the pharmacokinetics and metabolism of codeine in patients.     

Determination of trichlormethiazide in human plasma and urine by high-performance liquid chromatography

Summary This paper describes a high-performance liquid chromatographic (HPLC) assay method for the determination of trichlormethiazide (TCM) in human plasma and urine. After extraction and separation on an ODS column TCM from plasma was detected by oxidation in an electrochemical detector (ECD) by a porous graphite electrode. The sensitivity was better than HPLC with UV detection, enabling the determination of 2 ng ml^–1 TCM in human plasma. This method also allows determination of TCM at higher concentrations by exchanging the UV for the electrochemical detector. To study the pharmacokinetics, TCM in plasma and urine was assayed with coefficients of variation in the range 2–3%. The method has the advantages of high sensitivity for plasma assay and high precision with a simple procedure for both plasma and urine samples. Small samples of 0.5 ml plasma per assay also reduced the total volume of plasma needed.     

Development of superparamagnetic high-magnetization C18-functionalized magnetic silica nanoparticles as sorbents for enrichment and determination of methylprednisolone in rat plasma by high performance liquid chromatography

In this study, a novel extraction and enrichment technique based on superparamagnetic high-magnetization C₁₈-functionalized magnetic silica nanoparticles (C₁₈-MNPs) as sorbents was successfully developed for the determination of methylprednisolone (MP) in rat plasma by high performance liquid chromatography (HPLC). The synthesized silica-coated magnetite modified with chlorodimethyl-n-octadecylsilane was about 320 nm in diameter with strong magnetism and high surface area. It provided an efficient way for extraction and concentration of MP in the samples through hydrophobic interaction by the interior C₁₈ groups. Moreover, MP adsorbed with C₁₈-MNPs could be simply and rapidly isolated through placing a strong magnet on the bottom of container, and then easily eluted from C₁₈-MNPs by n-hexane solution. Extraction conditions such as amounts of C₁₈-MNPs added, adsorption time and desorption solvent, were investigated. Method validations including linear range, detection limit, precision, and recovery were also studied. The results showed that the proposed method based on C₁₈-MNPs was a simple, accurate and high efficient approach for the analysis of MP in the complex plasma samples.     

HPLC determination and pharmacokinetic studies of salvianolic acid B in rat plasma after oral administration of Radix Salviae Miltiorrhizae extract

A precise and reproducible HPLC method has been established and validated for determination of salvianolic acid B (SalB) in rat plasma after oral administration of Radix Salviae Miltiorrhizae extract. Liquid-liquid extraction was adopted for the sample preparation. Separation was accomplished on a C(18) column with a linear gradient elution consisting of acetonitrile and aqueous phosphoric acid. Ultraviolet detection was at 280 nm. The method was validated over the concentration range 10.8-259.4 microg/mL using 1 mL of plasma. The assay was linear over this concentration range with a coefficient of variation less than 7%. The extraction recovery of SalB was within the range 71-83% with RSD 11%. The mean recovery of the internal standard was 84% (n = 6) with RSD of 5.6%. This method is suitable to determine SalB in plasma and to investigate the pharmacokinetics of SalB.     

Determination of the retinoid (E)-1,2,3,4-tetrahydro-1,1,4,4-tetramethyl-6-(1-methyl-2- phenylethenyl)naphthalene and its phenolic metabolite in human plasma by reversed-phase high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic assay was developed for the determination in plasma of (E)-1,2,3,4-tetrahydro-1,1,4,4-tetramethyl-6-(1-methyl-2- phenylethenyl)naphthalene (1) and its phenolic metabolite (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2- methylethenyl]-phenol (2). An extraction procedure using protein precipitation and liquid-liquid extraction was combined with a simple column-switching technique. To minimize sample consumption, the assay was adapted to a sample volume of 200 microliters, which was sufficient for more than 90% of all determinations. The quantification limit was 100 ng/ml for 1 and 2, whereas the detection limits were 20 and 30 ng/ml, respectively. The recoveries for 1 and 2 were 91 and 94%, respectively, using ultraviolet detection at 280 nm. The assay was used to quantify both compounds in human plasma samples.     

Ultrasensitive method to quantify intracellular zidovudine mono‐, di‐ and triphosphate concentrations in peripheral blood mononuclear cells by liquid chromatography–tandem mass spectrometry

Although zidovudine (AZT) is not the preferred antiretroviral drug for adult HIV‐infected patients, it is still widely used in infants for both prevention of mother‐to‐infant HIV‐1 transmission and treatment of HIV‐infected children. However, it is difficult to measure intracellular concentrations of AZT metabolites in small blood samples due to their extremely low concentrations in peripheral blood mononuclear cells and interference by endogenous nucleotide triphosphates, residual plasma phosphates and electrolytes. We developed an ultrasensitive assay using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for measurement of intracellular concentrations of zidovudine (AZT)‐monophosphate (AZT‐MP), ‐diphosphate (AZT‐DP) and ‐triphosphate (AZT‐TP). The high sensitivity was due to the improvement of peripheral blood mononuclear cells extraction for complete removal of plasma and electrolytes, alkalization of LC buffer and use of alkaline‐stable high performance liquid chromatography column and tetrabutylammonium hydroxide as the ion pair. Using this method, the lower limits of quantification of AZT, AZT‐MP, ‐DP and ‐TP were 6, 6, 10 and 10 fmol per sample, respectively. Accuracy ranged 89–115% and precision was lower than 15% in the quantification range of 6–6000 fmol/sample for plasma AZT and intracellular AZT‐MP and 10–10 000 fmol/sample for AZT‐DP and ‐TP. The validation parameters met the international requirements. Among nine AZT‐treated HIV‐infected adult patients, five had low AZT‐TP levels (<10 fmol/10⁶ cells). Our assay has high sensitivity and is advantageous for evaluation of AZT phosphates in children and infants based on minimum blood sampling requirement. Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous determination of risperidone and 9-hydroxyrisperidone in plasma by liquid chromatography/electrospray tandem mass spectrometry

A simple and highly sensitive liquid chromatographic/electrospray tandem mass spectrometric (LC/MS/MS) assay was developed for the simultaneous determination of risperidone (RSP) and its major circulating metabolite 9-hydroxyrisperidone (9-OH-RSP) in the plasma of humans and rats. A simple one-step solvent extraction with 15% methylene chloride in pentane was used to isolate the compounds from plasma. The compounds were eluted from a phenyl-hexyl column and detected with a Perkin-Elmer SCIEX API2000 triple-quadrupole mass spectrometer using positive ion atmospheric pressure electrospray ionization and multiple reaction monitoring. The assay was linear over the range 0.1-100 ng ml(-1) when 0.5 ml of plasma was used in the extraction. The overall intra- (within-day) and inter- (between days) assay variations were < 11%. The variations in the concentrations of two long-term quality control samples from pooled patient plasma samples analyzed over a period of 6 months were approximately 10%. The analysis time for each sample was 4 min and more than 100 samples could be analyzed in one day by running the system overnight. The assay is simple, highly sensitive, selective, precise and fast. This method is being used for the therapeutic drug monitoring of schizophrenic patients treated with RSP and to study the pharmacokinetics and tissue distribution of RSP and 9-OH-RSP in rats.     

Determination of flurbiprofen enantiomers in plasma using a single-isomer amino cyclodextrin derivative in nonaqueous capillary electrophoresis

A nonaqueous capillary electrophoresis (NACE) assay was developed for the separation and determination of flurbiprofen enantiomers in plasma samples using 6-monodeoxy-6-mono(3-hydroxy)propylamino-beta-cyclodextrin as chiral selector. The nonaqueous background electrolyte was made up of 40 mM ammonium acetate in methanol (MeOH), and flufenamic acid was employed as internal standard. Solid-phase extraction was used for sample cleanup prior to the NACE separation. The NACE method reproducibility was optimized by evaluating different capillary washing sequences between runs. After having tested various conditions, trifluoroacetic acid (1 M) in MeOH was finally selected. Concerning the solid-phase extraction procedure, good and reproducible analyte recoveries were obtained using MeOH for protein denaturation and a polymeric phase combining hydrophobic interactions with anion exchange properties (Oasis) MAX) was selected as extraction sorbent. The method selectivity was not only demonstrated toward a blank plasma sample but also toward other non-steroidal anti-inflammatory drugs. The method was then successfully validated with respect to response function, trueness, precision, accuracy, linearity and limit of quantification.     

Simultaneous determination of methylprednisolone and methylprednisolone 21-[8-[methyl-(2-sulfoethyl)amino]-8-oxooctanoate] sodium salt in human urine by high-performance liquid chromatography with ultraviolet detection

A reversed-phase high-performance liquid chromatographic assay with ultraviolet detection at 243 nm has been developed for the quantitative determination of methylprednisolone (MP) and methylprednisolone 21-[8-[methyl-(2-sulfoethyl)amino]-8-oxooctanoate] sodium salt (MPSO) in human urine following therapeutic doses in humans. The assay procedure involves stabilization of urine samples by addition of disodium ethylenediaminetetraacetic acid (Na2EDTA) and ion-pair extractions of MPSO using tetraethylammonium chloride (TEACl) as the counter ion. After extracting both drugs and internal standard into chloroform, the extract was evaporated to dryness under nitrogen. The resulting residue was reconstituted in 200-500 microliters of mobile phase and chromatographed on an IBM C18 reversed-phase column (5 microns). The mobile phase was a mixture of water-acetonitrile-isopropanol (71.2:18.8:10.0, v/v) containing 75 microliters of 0.1 M hydrochloric acid and 0.450 g of TEACl per liter. Propyl p-hydroxybenzoate was used as an internal standard. The extraction efficiencies of MP and MPSO were greater than 90% using the ion-pairing agent TEACl. The chromatographic responses were linear up to about 200 micrograms/ml for MP and 80 micrograms/ml for MPSO and had sufficient precision and accuracy to provide quantitative data from human urine. The assay detection limit was about 8 ng/ml for MP and 25 ng/ml for MPSO in human urine. Stability studies in urine indicated that without Na2EDTA stabilization and at room temperature, rapid degradation of MPSO occurred in urine. Addition of EDTA to the urine specimen and storage at -70 degrees C increased the stability of MPSO, and little or no degradation was observed in urine stored for more than 60 days. The method has been used in the simultaneous determination of MP and MPSO in urine specimens obtained from a single-dose tolerance study of MPSO in normal male volunteers.     

Determination of Methyl Parathion in Water and Its Removal on Zirconia Using Optical Enzyme Assay

A simple, miniaturized microplate chemiluminescence assay for determination of methyl parathion (MP) was developed in 384-microwell plates. Zirconia (ZrO₂) was added in microwell for adsorption of acetylcholinesterase (AChE). The developed assay is based on inhibition of AChE by MP. A good dynamic range 0.008–1,000 ng/mL was obtained for MP with limit of detection 0.008 ng/mL. Intrabatch and interbatch reproducibility for miniaturized assay was obtained with % RSD up to 3.07 and 5.66, respectively. In 384 well plate formats, 70 samples were simultaneously analyzed within 20 min with assay volume of 41.5 μL. The application of developed assay was extended for MP remediation. Column containing ZrO₂ was utilized for remediation where MP was selectively adsorbed. Under optimized condition, adsorption of MP on ZrO₂ was found to be 98–99% with 2-h contact time in real water samples. Adsorption of MP on ZrO₂ column followed by quantification using developed bioassay provides a novel approach to monitor remediation. The applicability of assay was successfully extended for determination of MP in water samples after removal through ZrO₂.     

Determination of cibenzoline in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of cibenzoline (CipralanTM) in human plasma and urine. The assay involves the extraction of the compound into benzene from plasma or urine buffered to pH 11 and HPLC analysis of the residue dissolved in acetonitrile-phosphate buffer (0.015 mol/l, pH 6.0) (80:20). A 10-microns ion-exchange (sulfonate) column was used with acetonitrile-phosphate buffer (0.015 mol/l, pH 6.0) (80:20) as the mobile phase. UV detection at 214 nm was used for quantitation with the di-p-methyl analogue of cibenzoline as the internal standard. The recovery of cibenzoline in the assay ranged from 60 to 70% and was validated in human plasma and urine in the concentration range of 10-1000 ng/ml and 50-5000 ng/ml, respectively. A normal-phase HPLC assay was developed for the determination of the imidazole metabolite of cibenzoline. The assays were applied to the determination of plasma and urine concentrations of cibenzoline and trace amounts of its imidazole metabolite following oral administration of cibenzoline succinate to two human subjects.     

Determination of 2,3-benzodiazepine derivatives in rat plasma by high-performance liquid chromatography.

A simple high-performance liquid chromatographic method with ultraviolet detection at 240 nm for determination of a novel AMPA/kainate antagonist 1-(4'-aminophenyl)-3,5-dihydro-7,8-dimethoxy-2,3-benzodiazepine (2,3-BZ 6), and its derivatives in rat plasma is described. The procedure involves a fast extraction of the drugs from the plasma spiked with an internal standard. The samples are applied to a pre-packed glass column and drugs are eluted using ethyl acetate. A linear response was observed over the examined concentration range. The lower limit of detection of 2,3-BZ 6 was 5.5 ng/ml. The assay has been used to determine the time course of plasma levels of the 2,3-benzodiazepine derivatives in Sprague-Dawley rats.     

Determination of methamphetamine and amphetamine in abusers' plasma and hair samples with HPLC-FL

This paper describes highly sensitive HPLC methods for the determination of amphetamine (AP) and methamphetamine (MP) in abusers' plasma and hair samples. AP and MP were derivatized with the fluorescent reagent, DIB-Cl, to yield a highly fluorescent DIB-derivatives of AP and MP, which were then analyzed by HPLC with fluorescence detection at excitation and emission wavelengths of 325 and 430 nm, respectively. The separation was achieved on an ODS column with isocratic mobile phases composed of acetoniltrile and citrate buffer (55:45, v/v) for plasma samples and of acetonitrile-methanol-citrate buffer (45:20:37.5, v/v/v) for hair samples. The limits of detection were less than 0.87 ng/mL and 0.12 ng/mg in plasma and hair samples, respectively, for both AP and MP. The methods were then applied to the determination of MP and its metabolite AP in plasma obtained from two cases of illegally ingested MP and in one of the cases' hair received later. Case I was treated with dialysis; samples before and after dialysis were analyzed by the described method. After dialysis for 5 h, the total plasma levels of AP and MP decreased from 720 to 190 ng/mL. For case II, MP and AP levels were monitored for 3 days after digestion. Total plasma levels decreased from 57 ng/mL in the day of digestion to 11 ng/mL after 3 days. In hair samples, AP and MP could also be detected in very low concentrations.     

A high-performance liquid-chromatographic method for the determination of ochratoxin a in human plasma

Summary A high-performance liquid-chromatographic method is described for the quantitative determination of the mycotoxin ochratoxin A (OTA) in human plasma. The assay involves extraction with chloroform and sodium bicarbonate then HPLC with fluorescence detection. The method was validated in terms of selectivity, recovery, linearity, precision (within-day and between-day variability), accuracy, detection and quantification limits, and the stability of OTA in plasma and treated samples. The limit of detection was 0.4 ng mL⁻¹ of OTA in methanol, corresponding to 0.52 ng ml⁻¹ OTA in plasma. This assay was successfully applied for the determination of OTA levels in human plasma.     

Determination of Chlordiazepoxide and Its Metabolites in Plasma by High Pressure Liquid Chromatography

Abstract

A rapid, sensitive and specific high pressure liquid chromatographic (HPLC) assay was developed for the determination of chlordiazepoxide and its metabolites from plasma. The assay involves extraction of chlordiazepoxide and its metabolites into diethyl ether from plasma buffered to pH 9. The overall recovery of chlordiazepoxide is 80 ± 5.0% (S.D.) and the sensitivity limit of detection is 50 to 100 ng/ml of plasma, using a 1 ml specimen. The assay was used in the determination of plasma levels of chlordiazepoxide and its metabolites in man following oral administration of chlordiazepoxide. HCl.

The chromatographic behavior of other clinically important benzodiazepines and their major metabolites is also reported.     

Simultaneous determination of losartan,EXP-3174 and hydrochlorothiazide in plasma via fully automated 96-well-format-based solid-phase extraction and liquid chromatography–negative electrospray tandem mass spectrometry

An automated, sensitive and high-throughput liquid chromatographic/electrospray tandem mass spectrometric (LC–MS/MS) assay was developed for the simultaneous determination of losartan (LOS), its major circulating metabolite EXP-3174 and hydrochlorothiazide (HCTZ) in human plasma. LOS and HCTZ coexist in the same drug formulation, and this is the first method that enables the simultaneous determination of both drugs along with the active metabolite of LOS. Since these drugs have different physicochemical properties, the employment of a liquid–liquid extraction (LLE) protocol was precluded. A fully automated solid-phase extraction (SPE) protocol, based on 96-well format plates, was used to isolate these compounds and furosemide (internal standard, IS) from plasma. Washing and elution steps were amended accordingly in order to minimize any matrix effect from components of the plasma without reducing the elution of the molecules of interest. The compounds were eluted from a C18 column and detected with an API 3000 triple-quadrupole mass spectrometer using negative electrospray ionization and multiple reaction monitoring (MRM). The assay was linear over the range 1.00–400 ng/mL for LOS and EXP-3174 and 0.500–200 ng/mL for HCTZ, respectively, when 200 μl of plasma was used in the extraction. The overall intra- and interassay variations were within acceptance limits. The analysis time for each sample was 4 min, and more than 300 samples could be analyzed in one day by running the system overnight. The assay was simple, highly sensitive, selective, precise, fast, and it enables the reliable determination of LOS, EXP-3174 and HCTZ in pharmacokinetic or bioequivalence studies after per os administration of a single tablet containing both drugs.     

Application of multiwalled carbon nanotubes for solid-phase extraction of organophosphate pesticide

Multiwalled carbon nanotube (MWCNT) was developed as a new sorbent for solid-phase extraction (SPE) of organophosphate (OP) pesticides. A combination of SPE with square-wave voltammetric (SWV) analysis resulted in a fast, sensitive, and selective electrochemical method for determination of OP pesticide using methyl parathion (MP) as a representative. Because of the strong affinity of MWCNT for phosphoric group, nitroaromatic OP compounds can strongly bind to the MWCNT surface. The macroporosity and heterogeneity of MWCNT allow extracting a large amount of MP less than 5 min. The stripping response was highly linear over the MP range of 0.05–2.0 μg/mL, with a detection limit of 0.005 μg/mL. The determination of MP in garlic samples showed acceptable accuracy. The fast extraction ability of MWCNT makes it promising sorbent for various solid-phase extractions.     

Determination of vinca alkaloids in plasma and urine using ion-exchange chromatography on silica gel and fluorescence detection

The development of a method for the determination of the antineoplastic vinca alkaloids vinblastine and vindesine in biological samples is described. The selectivity of the assay is high owing to the use of solid-phase extraction on a cyanopropyl extraction column prior to isocratic chromatography on unmodified silica gel with fluorescence detection. The influence of acetonitrile concentration and mobile phase pH on the capacity factors of the drugs was studied in order to optimize the separation between the drugs and endogenous components. The effect of varying the type and concentration of competing cations in the mobile phase was also examined. The limit of determination (signal-to-noise ratio = 3) for vinblastine is 0.5 ng/ml in plasma and urine and for vindesine 2.5 ng/ml. The assay is suitable for determining the concentrations of both compounds in plasma and urine samples from patients.