Formic Acid In Methanol Research Articles

Facile separation of four co-formulated ternary antihypertensive drug combinations with a customized elution protocol using supercritical fluid chromatography

A novel supercritical fluid chromatography (SFC) method is described to analyze four antihypertensive drug combinations containing a diuretic (hydrochlorothiazide, HCT), a calcium channel blocker (amlodipine, AML) and an angiotensin receptor blocker (olmesartan (OLM)/ telmisartan (TEL)/valsartan (VAL)/renin inhibitor (aliskiren, ALI) using a customized protocol. The effect of mobile phase, analytical column, temperature and pressure were suitably optimized. The best conditions for separation was achieved on DCPak P4VP (250 × 4.6 mm, 5 µm) column using CO2 and 0.1% formic acid in methanol by gradient elution for all drug combinations. The chromatographic separations were accomplished within 5.0 min at 125 bar back pressure and 40 °C column temperature. Detection and quantitation of analytes was performed at 230 and 250 nm using a photo diode array detector. The resolution factor Rs between the drugs ranged from 1.52 to 3.85 for different combinations. The study showed decrease in retention factors (k) of the drugs with increase in temperature and pressure. The van’t Hoff plots revealed enthalpy driven process for the separation of all drug combinations. The method was validated as per ICH guidelines for specificity, sensitivity, accuracy and precision and recovery. Method linearity was established from 10.0–300, 1.0–20/25, 2.00–100, 5.00–200, 2.00–100 and 5.00–200 µg/mL for ALI, AML, HCT, OLM, TEL and VAL, respectively. Application of the method was demonstrated by analyzing single pill combinations of ALI–AML–HCT, OLM–AML–HCT, TEL–AML–HCT and VAL–AML–HCT. The significance of the method for accuracy and precision was assessed using Student’s t-test and F-test.

Polar Reversed-Phase Liquid Chromatography Coupled with Triple Quadrupole Mass Spectrometer Method for Simple and Rapid Determination of Maleic Hydrazide Residues in some Fruits and Vegetables

Maleic hydrazide (MH) is used as a plant growth regulator, herbicide, and sprouting inhibitor for some crops to improve the growth and the quality of some fruits and vegetables so that MH residues should be analyzed in food. Most of the chromatographic analysis methods for MH residues were operated by using ion chromatographic (IC) columns which need complicated with extra washing and conditioning steps. The developed method has overcome this problem by using Synergi polar reversed-phase (RP) column and non-scheduled multiple-reactions monitoring (MRM) scanning modes. Liquid chromatography coupled with negative electrospray ionization triple quadrupole mass spectrometer LC-ESI (-) MS/MS method was developed and validated according to SANTE/12682/2019 at four fortification levels in four food matrices. The method optimization was carried out by using different extracting solvents, LC columns, and mobile phase ratios. The extraction was developed and operated by using 1% formic acid in methanol. The limits of quantification (LOQ) for MH were in the range 0.05–0.1 μg/g that was below the EU maximum residue limit (MRL), set for the studied food matrices such as onion, potatoes, citrus, and grape (15, 50, 0.2, and 0.2 μg/g) respectively. Good method linearity was obtained in the concentration range 0.01–2 μg/ml with correlation coefficients (r2) ≥ 0.99. The average recoveries ranged from 84 to 110% with relative standard deviations RSDs ≤ 17%. The matrix effects on the MH signals were studied and compensated by using the standard addition technique. This polar-RP-LC-ESI (-) MS/MS method was developed to help laboratories which determine the MH residues in food.

LC-MS-MS Determination of 25 Antipsychotic Drugs and Metabolites in Urine for Medication Compliance Monitoring.

A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed for 25 antipsychotic drugs and their metabolite in urine for monitoring medication compliance of mentally disordered criminals on probation. Target compounds were extracted with a solid-phase extraction technique using a newly developed hydrophilic-lipophilic balanced sorbent to remove interferences and minimize the matrix effect (ME). Extracted sample was injected into the LC-MS-MS with an electrospray ionization source in positive mode and multiple-reaction monitoring mode. The analytes were separated and detected within 10 minutes using a reversed-phase column with a gradient elution method using 0.1% formic acid in water and 0.1% formic acid in methanol as mobile phase. The validation parameters were evaluated as follows: selectivity, limit of detection, lower limit of quantification (LLOQ), linearity, accuracy and precision, stability, dilution integrity, recovery (RE), ME and process efficiency (PE). The LLOQs were 0.1 to 2.0 ng/mL, and determination coefficients of the calibration curve were above 0.9943 over the concentration ranges. The intra-and inter-day accuracy ranged from -10.4% to 9.9% and from -9.6% to 9.4%, while the intra-and inter-day precision were within 10.7% and 9.9%. The bench-top and long-term stability ranged from 92.1% to 109.5% and 88.7% to 111.6%, respectively. The reproducibility of auto-sampler stability was <10% for all analytes. The accuracy and precision of dilution integrity ranged from -11.7% to 10.5% and 0.4% to 9.9%, respectively. The relative standard deviation of RE and ME was from 0.6% to 6.6% and 0.5% to 3.9%, respectively, while that of PE was 1.3% to 4.5%. The developed LC-MS-MS method for medication compliance monitoring was successfully applied to urine samples from mentally disordered probationers and determined to be one of effective ways for preventing the recurrence of mentally disordered crimes.

Simultaneous quantitation of four androgens and 17-hydroxyprogesterone in polycystic ovarian syndrome patients by LC-MS/MS.

BackgroundDue to the low concentration of androgens in women and the limitation of immunoassays, it remains a challenge to accurately determine the levels of serum androgens in polycystic ovary syndrome (PCOS) patients for clinical laboratories. In this report, a liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method was developed and validated for simultaneous quantitation of testosterone (T), androstenedione (A4), dehydroepiandrosterone sulfate (DHEAS), dihydrotestosterone (DHT), and 17‐hydroxyprogesterone (17‐OHP) that are associated with PCOS.MethodsThe serum samples were processed by protein precipitation and solid phase extraction before analysis with the in‐house developed LC‐MS/MS. The chromatographic separation was achieved with a C18 column, using a linear gradient elution with two mobile phases: 0.02% formic acid in water (phase A) and 0.1% formic acid in methanol (phase B). The separated analytes were detected by positive or negative electrospray ionization mode under multiple reaction monitoring (MRM).ResultsThe assay for all the five analytes was linear, stable, with imprecision less than 9% and recoveries within ±10%. The lower limits of quantification were 0.05, 0.05, 5, 0.025, and 0.025 ng/mL for T, A4, DHEAS, DHT, and 17‐OHP, respectively. In the receiver operating characteristic curve (ROC) analyses with the PCOS (n = 63) and healthy (n = 161) subjects, the AUC of the four‐androgen combined was greater than that of any single androgen tested in PCOS diagnosis.ConclusionsThe LC‐MS/MS method for the four androgens and 17‐OHP showed good performance for clinical implementation. More importantly, simultaneous quantitation of the four androgens provided better diagnostic power for PCOS.

Simultaneous separation and determination of 32 fentanyl-related substances, including seven sets of isomeric fentanyl analogues, by ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry.

A method for separation and determination of 32 fentanyl-related substances, including seven sets of isomeric fentanyl analogues, was developed using ultra-high-performance liquid chromatography coupled with quadrupole-orbitrap high-resolution mass spectrometry. The collision energy, chromatographic column, and mobile phase were optimized. All compounds were efficiently flushed out of a universal C18 column with a soft gradient consisting of solvent A (2mM ammonium formate and 0.1% formic acid in water) and solvent B (2mM ammonium formate and 0.1% formic acid in methanol) in only 20 min, achieving excellent resolution. Detection and analysis were carried out simultaneously in the positive ion mode using the full scan and data-dependent tandem mass spectrometry modes with a normalized collision energy of 40. The method was validated in terms of limit of detection, limit of quantification, linearity, accuracy, and precision. For all fentanyl-related substances, the limit of detection (0.5ng/mL) and limit of quantification (1ng/mL) were adequate for screening and quantification in daily drug control. Calibration curves for all compounds were established in the range of 1-500ng/mL. The intra- and interday precision (RSD%) were within 0.4-2.3 and 0.7-2.7%, respectively. The accuracy ranged from 99 to 106%. The method was applied to analyze seized drug samples.

Simultaneous determination of podophyllotoxin, quercetin and kaempferol in podophyllin by liquid chromatography tandem mass spectrometry

A quantitative high performance liquid chromatography-tandem mass spectrometry (LC/MS/MS) was developed to analyze the constituents of podophyllin. Chromatographic separation was performed on a Cosmosil 5C18-MS (25 cm×4.6 mm I.D., 5 μm) reverse phase column using a gradient of mobile phase (0.25% formic acid-methanol). The column effluent was split 2:3 into the photodiode detector and tandem mass spectrometer. Podophyllotoxin, quercetin and kaempferol in podophyllin were identified by daughter ion scan mode and then determined their contents by multiple-reaction monitoring (MRM) mode. The limits of detection and quantitation for podophyllotoxin, quercetin and kaempferol were 2.40, 8.01; 2.94, 9.87 and 3.10, 10.2 ng/mL, respectively. The relative standard deviations of intraday and interday analyses for podophyllotoxin, quercetin and kaempferol ranged from 0.52-6.01% and 2.48-9.88%, respectively. The mean recoveries for podophyllotoxin, quercetin and kaempferol were 101.4%, 98.3% and 98.7%, respectively. The developed LC/MS/MS method was suitable for the simultaneous determinations of podophyllotoxin, quercetin and kaempferol in podophyllin.

Determination of free and total available ferulic acid in different types of Chinese Angelica by high performance liquid chromatography

Free and conjugated forms of ferulic acid (FA) are generally available in higher plant taxa such as Chinese Angelica (CA, the roots of Angelica sinensis (Oliv.) Diels). These various forms of FA were found readily inter-convertible and the extractable level of each depended on solvent properties and acidity. Extraction efficiency using various pH solutions namely, water, 70% methanol, methanol-formic acid (95:5) and methanol-2% NaHCO3 in water (95:5) was compared. Extractable FA were found varying in samples under neutral solvents extraction, whilst relatively consistent for slightly acidic and alkali solvents which were therefore chosen as the optimal media to extract and determine the reproducible levels of free and total available FA. An accurate and rapid high performance liquid chromatographic (HPLC) analysis was conducted using an Alltima C18 column (5 μm, 4.6 mm i.d. × 250 mm) with a guard column (C18, 5 μm, 4.6 mm i.d. × 7.5 mm) at 30°C, eluted with a mixture of 1.0% acetic acid and acetonitrile in a gradient program at a flow rate of 1.0 mL/min and detected at 320 nm. Altogether 20 different types of CA samples including whole root, root head, rootlets, whole root slice, Angelica processed by Chinese yellow wine, and charred Angelica were quantified for free and total available FA. Total available FA was found more abundant than free counterpart with an average ratio of 3.15 (n = 20) in the range of 1.29 to 8.23 for these CA samples. The extraction protocol was proven reliable to quantitatively convert all conjugated FA into its free forms and thereby accurately determined by HPLC method for quality assessment.

Quantitative analysis of caffeine, ethoxybenzamide, chlorzoxazone, diazepam and indomethacine illegally adulterated in Chinese medical pills

A traditional Chinese medicine, which was brought by a consumer for treating heart disease and uricosuria, was obtained and tested the illegal adulteration with chemical drugs. In the qualitative analysis, two-dimensional TLC, with chloroform:ethanol (9:1,v/v) and ethyl acetate: methanol: ammonia water (8:1:1,v/v) as the solvent systems, established the presence of caffeine, indomethacin, ethoxybenzamide, chlorzoxazone and diazepam in the pills. For quantitative analysis, a reversed-phase HPLC method was established for simultaneous determination of the five chemical drugs. The analysis of the sample extract was performed on a Cosmosil 5C 18-AR column (4.6x150mm) by a linear gradient elution with the mobile phase of 0.05 M KH 2 PO 4-0.1% formic acid buffer and methanol, programmed from 60:40, v/v, to 40:60, v/v, for 30 min, with 30 mim hold and then 60:40, v/v, for the subsequent 15 min, at a flow rate of 0.7 ml/min and with propylparaben as an internal standard. The monitor wavelength was set at 230 nm. System suitability tests showed that the established method was quite suitable for the determination of the adulterants in the pills. By using this method, 30.8mg of caffeine, 71.4 mg of ethoxybenzamide, 35.7 mg of chlorzoxazone, 0.6 mg of diazepam and 9.3 mg of indomethacin, were found in each pill of the sample.

The determination of β‐agonist residues in bovine tissues using liquid chromatography–tandem mass spectrometry

We aimed to develop a rapid, simple and reproducible method based on LC-tandem mass spectrometry (LC-MS/MS) to analyze β-agonist residues (clenbuterol, zilpaterol, ractopamine and isoxsuprine) in bovine tissues. The method was validated in accordance with the European Council Decision 2002/657/EC. The samples were homogenized, and then 10 mL of an acetate buffer was added to a 5-g sample. The sample was then centrifuged at 12,000 rpm and filtered. Sodium hydroxide (2 m) was added to adjust pH of the sample that was centrifuged again. The extract was filtered through a solid-phase extraction column. The residue was re-dissolved in 250 μL acetonitrile and then subjected to LC-MS/MS. The separation was done on a C18 column. The mobile phase consisted of 0.1% formic acid in deionized water and 0.1% formic acid in methanol. The mean recoveries of β-agonists were in the range of 84.3%-119.1% with relative standard deviations (%RSDs) of 0.683%-4.05%. Decision limits and detection capabilities of the analytes ranged from 0.0960 to 4.9349 μg/kg and from 0.0983 to 5.0715, respectively. This method was used to detect four β-agonists in 100 bovine muscle, 100 liver and 100 kidney tissues from a slaughterhouse. No residue was found above the maximum residue limit level.

Development of UPLC-MS/MS method for analyzing phorate: application to wastewater treatment

A sensitive and selective assay based on liquid chromatography–tandem mass spectrometric method was developed for quantification of phorate in river water. The chromatographic separation of phorate was achieved on Zorbax C18 column using two mobile phase consisting 0.1% formic acid in methanol, and 0.1% formic acid in MilliQ water (v/v ratio of 96:04) by isocratic elution method at a flow rate of 300 µL/min. The quantification was carried out using multiple reaction monitoring transitions of m/z 261/75 for phorate. The lower limit of detection and limit of quantification for the developed UPLC-MS/MS was 0.08 ng/mL and of 0.19 ng/mL respectively. The assay was validated for selectivity, linearity, accuracy and precision. The developed assay was then employed for quantification of phorate in river water and wetland treated water. The role of constructed wetlands (CW) in phorate removal was established in this study. The removal efficiency of phorate by Constructed Wetland was found to be 60–90% along with an overall 70–90% reduction in chemical oxygen demand (COD), biological oxygen demand (BOD) and phosphorus (P).

Factorial Design Optimisation of Solid Phase Extraction for Preconcentration of Parabens in Wastewater Using Ultra-High Performance Liquid Chromatography Triple Quadrupole Mass Spectrometry

Background: Parabens are synthetic esters used extensively as preservatives and/or bactericides in personal care personal products. Objective: Development and validation of a novel robust chemometric assisted analytical technique with superior analytical performances for the determination of ethylparaben, methylparaben and propylparaben, using simulated wastewater matrix. Methods: An automated Solid Phase Extraction (SPE) method coupled with liquid chromatographymass spectrometry was applied in this study. A gradient elution programme comprising of 0.1% formic acid in deionised water (A) and 0.1% formic acid in Methanol (B) was employed on a 100 x 2.1 mm, 3.0 μm a particle size biphenyl column. Two-level (2k) full factorial design coupled with response surface methodology was used for optimisation and investigation of SPE experimental variables that had the most significant outcome of the analytical response. Results: According to the analysis of variance (ANOVA), sample pH and eluent volume were statistically the most significant parameters. The method developed was validated for accuracy, precision, Limits of Detection (LOD) and Limit of Quantification (LOQ) and linearity. The LOD and LOQ established under those optimised conditions varied between 0.04-0.12 μgL−1 and 0.14-0.40 μgL−1 respectively. The use of matrix-matched external calibration provided extraction recoveries between 78-128% with relative standard deviations at 2-11% for two spike levels (10 and 100 μgL-1) in three different water matrices (simulated wastewater, influent and effluent water). Conclusion: The newly developed method was applied successfully to the analyses of parabens in wastewater samples at different sampling points of a wastewater treatment plant, revealing concentrations of up to 3 μgL−1.

Role of TLC to ensure manifestation of individual ingredients in herbal formulation

Herbal medicine is gaining its popularity day by day and gradually streaming toward integration into the mainstream healthcare systems. Therefore, standardization of herbal formulations is essential in order to assess quality of the drugs. Two commonly used paradigms, compound and pattern-based approaches, were performed to ensure quality and hence product consistency. A rapid, sensitive, and reproducible high-performance thin-layer chromatography (HPTLC) method has been developed for one of the major bioactive markers, piperine. The plates were developed using the mobile phase with toluene–ethyl acetate–formic acid–methanol (3:3:0.8:0.2, v/v). The method was validated for limit of detection, limit of quantification, linearity, specificity, precession, and recovery. The results indicate that the bioactive compound was present in 2.64% w/w in the fruit of Piper nigrum Linn. and 0.253% in a herbal formulation. Each ingredient was analyzed vis-a-vis the herbal formulation and the reflectance spectrum of the matching component(s) to ensure the presence of each ingredient in the herbal formulation. The individual component adding up has also resulted similar fingerprint pattern as that of herbal formulation. Thus, the TLC method could be a rapid tool for the delineation of each individual ingredient in the finished products for authenticity, efficacy, and for batch-to-batch consistency.

Estimation of Levodopa in the Unani Drug Mucuna pruriens Bak. and Its Marketed Formulation by High-Performance Thin-Layer Chromatographic Technique.

Mucuna pruriens Bak. Syn. Mucuna prurita Hook. seed is the rich source of levodopa (L-dopa) and has been used in traditional medicines to treat diseases resembling Parkinson's disease. In the present study, a new HPTLC method was developed and validated for estimation of L-dopa from M. pruriens (black- and white-colored seeds) collected from two different locations in India. TLC aluminum plates precoated with silica gel were used as the stationary phase. The plates were developed to a distance of 60 mm at a temperature of 22 ± 4°C in a twin glass chamber saturated with ethyl acetate-methanol-formic acid-water (15+3+3+4.5, v/v/v/v), as the mobile phase. The Rf value of L-dopa was found to be 0.45. L-dopa was quantified at 282 nm, the wavelength of maximum absorbance by a densitometric scanner. The TLC plate was derivatized by ninhydrin reagent and photodocumented. L-dopa showed a good linearity in the concentration range 400-1000 ng/spot. The linear regression analysis of calibration plot showed good linear relationship between peak area and peak height (r2 = 0.997). The method was validated for precision, repeatability, and accuracy. Recovery was determined by spiking L-dopa with samples and was found to be 96.10%. L-dopa was found to be present at concentration 3.02-4.72% in samples and its formulation.

Liquid chromatography-high resolution mass spectrometry for the determination of three cannabinoids, two (−)-trans-Δ9-tetrahydrocannabinol metabolites, and six amphetamine-type stimulants in human hair

Since cannabis and amphetamine-type stimulants (ATS) are drugs of abuse used alone and concurrently worldwide, it is important to analyze them simultaneously. However, there are no reports of analytical method for the simultaneous extraction of these compounds and metabolites in hair samples of suspected drug abusers, due to the different chemical properties of acidic and lipophilic cannabinoids, and basic and hydrophilic ATS. We developed a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method for the quantification of cannabidiol (CBD), cannabinol (CBN), (−)-trans-Δ9-tetrahydrocannabinol (THC), THC metabolites such as 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) and THC-COOH-glucuronide (THC-COOH-glu), and six ATS of amphetamine, methamphetamine, phentermine, methylenedioxyamphetamine, methylenedioxymethamphetamine, and methylenedioxyethylamphetamine in the least amounts of human hair samples. The pulverized hair samples (10 mg) were extracted with 1 mL of 0.5% formic acid in methanol three times, and the supernatants were evaporated in a stream of nitrogen gas. The residue was dissolved and the aliquot was analyzed by LC-HRMS using positive electrospray ionization and the parallel reaction monitoring mode. The lower limits of quantitation were 0.1 pg mg−1 for THC-COOH and THC-COOH-glu, 4 pg mg−1 for CBD, CBN, and THC, and 10 pg mg−1 for the six ATS. Linearity, accuracy, precision, matrix effect, recovery, and post-preparation stability for the 11 analytes were fully validated. The present method was successfully applied to the determination of 11 analytes in hair samples of 10 suspected drug abusers.

Effective PtAu nanowire network catalysts with ultralow Pt content for formic acid oxidation and methanol oxidation

Pt is the ideal anode catalyst in fuel cells. In this paper, in order to increase the utilization of Pt, the PtAu nanowire networks (NWNs) with ultralow content of Pt are fabricated by a simple silicon monoxide (SiO) reduction method without any capping agent. PtAu NWNs supported on carbon black with Pt content of 1 wt% (Pt0.05Au NWNs) are employed as catalysts for formic acid oxidation (FAO) and methanol oxidation reaction (MOR), whose mass activities are as high as 4998.9 and 2282.3 mA∙mgPt−1, respectively. The network structure facilitates the electron transfer and increases the stability of the catalysts. The PtAu composite experiences compressive lattice strain as confirmed by X-ray powder diffraction (XRD). The Pt0.05Au NWNs catalyst with low Pt content results in the largest strain variation compared with PtAu composited of other ratios, which may downshift the d-band center of Pt and lead to the higher electrocatalytic activity in oxidation reaction.

A rapid liquid chromatography/tandem mass spectrometry method for simultaneous determination of levodopa, carbidopa, entacapone and their six related compounds in film-coated tablets.

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated to determine levodopa, carbidopa, entacapone, and corresponding six related substances - levodopa impurity B, levodopa impurity C, methyldopa, methylcarbidopa, entacapone impurity C, and entacapone impurity A - in film-coated tablets for the first time. Chromatographic separation was achieved with a gradient elution by using a C18 column, a mobile phase containing 0.5% formic acid in water and 0.5% formic acid in methanol. The mobile phase flow rate was 0.5 mL min-1 . The UV detector was set at 280 nm and the triple quadrupole mass spectrometer was used in multiple reaction monitoring (MRM) mode. The limit of detection (LOD) and limit of quantification (LOQ) results were 1.3 and 3.94 ng mL-1 for levodopa impurity B; 5.26 and 15.9 ng mL-1 for levodopa impurity C; 0.833 and 2.53 ng mL-1 for methyldopa; 3.31 and 10.0 ng mL-1 for methylcarbidopa; 1.67 and 5.06 ng mL-1 for entacapone impurity C; and 0.61 and 1.86 ng mL-1 for entacapone impurity A. The method was rapid, linear, accurate, and reproducible. The LC/MS/MS method that was developed to determine the related substances and assay of levodopa, carbidopa, and entacapone can be used to evaluate the quality of regular samples in the pharmaceutical industry. It can be also used to test the stability of samples.

Quantification of Plasma and Urine Thymidine and 2’-Deoxyuridine by LC-MS/MS for the Pharmacodynamic Evaluation of Erythrocyte Encapsulated Thymidine Phosphorylase in Patients with Mitochondrial Neurogastrointestinal Encephalomyopathy

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an ultra-rare disorder caused by mutations in TYMP, leading to a deficiency in thymidine phosphorylase and a subsequent systemic accumulation of thymidine and 2’-deoxyuridine. Erythrocyte-encapsulated thymidine phosphorylase (EE-TP) is under clinical development as an enzyme replacement therapy for MNGIE. Bioanalytical methods were developed according to regulatory guidelines for the quantification of thymidine and 2’-deoxyuridine in plasma and urine using liquid chromatography-tandem mass spectrometry (LC–MS/MS) for supporting the pharmacodynamic evaluation of EE-TP. Samples were deproteinized with 5% perchloric acid (v/v) and the supernatants analyzed using a Hypercarb column (30 × 2.1 mm, 3 µm), with mobile phases of 0.1% formic acid in methanol and 0.1% formic acid in deionized water. Detection was conducted using an ion-spray interface running in positive mode. Isotopically labelled thymidine and 2’-deoxyuridine were used as internal standards. Calibration curves for both metabolites showed linearity (r > 0.99) in the concentration ranges of 10–10,000 ng/mL for plasma, and 1–50 µg/mL for urine, with method analytical performances within the acceptable criteria for quality control samples. The plasma method was successfully applied to the diagnosis of two patients with MNGIE and the quantification of plasma metabolites in three patients treated with EE-TP.

Simultaneous Determination of α-Glucosidase Inhibitory Triterpenoids in Psidium guajava Using HPLC-DAD-ELSD and Pressurized Liquid Extraction.

Psidium guajava, a popular food and medicine dual purposes plant cultivated in tropical and subtropical regions, has been widely used as food crop and folk medicine, such as anti-diabetes agent, around the world. Triterpenoids have been considered as the major active ingredients of P. guajava. In the present study, a high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors (HPLC–DAD–ELSD) method was developed for simultaneous determination of nine triterpenoids in P. guajava. Pressurized liquid extraction (PLE) was performed for sample preparation, and the analysis was achieved on a Cosmosil 5C18-MS-II (Nacalai Tesque, Kyoto, Japan) column eluted with gradient 0.1% aqueous formic acid-methanol system. The drift tube temperature of ELSD was set at 40 °C, and nitrogen flow-rate was at 1.6 L/min. All calibration curves for the analytes showed good linear regression (R2 > 0.9992) within test ranges. The established method was validated for intra-day and inter-day precisions (RSDs < 5%) and accuracy (recovery 94.23–106.87%). The validated method was successfully applied to determinate nine triterpenoids in 15 samples from the leave or fruit of P. guajava. In addition, the α-glucosidase inhibition assay showed good α-glucosidase inhibition activity in almost all the determined triterpenoids. The present study suggested that triterpenoids should be the quality control markers for P. guajava and HPLC–DAD–ELSD was an effective tool for the quality control of P. guajava.

Quantification of plasma levels of antiviral drug sofosbuvir and its metabolite GS331007 in patients of chronic hepatitis C with chronic kidney disease using UPLC-MS/MS method

Sofosbuvir based regimens are the only treatment available in India and some other Asian countries for curing hepatitis C viral (HCV) infection. The main excretion route of sofosbuvir is renal so treatment of HCV infection is challenging in patients on hemodialysis. A simple and sensitive UPLC-MS/MS method was developed and validated according to USFDA guidelines on Linear Ion trap Quadrupole tandem mass spectrometer (QTRAP4500) which was applied to estimate the drug concentration of sofosbuvir and its metabolite GS331007 in patients of chronic kidney disease with HCV infection. Clopidogrel was used as internal standard (IS) for this study. All analytes and IS were separated on UPLC C18-HSS column (2.1mmX50mm, 1.7μm) with retention time of 2.07, 0.29 and 1.58 min, respectively, by using mobile phases of 5mM ammonium acetate in 0.2% formic acid in water (A) and 5mM ammonium acetate in 0.2% formic acid in methanol (B) on gradient elution mode at a flow rate of 0.6 mL/min. Calibration curve was plotted over the range of 10-200 μg/mL for both of the analytes and equation was calculated by applying linear regression method. Detections of daughter ions (sofosbuvir-530 to 243m/z, GS331007-261 to 112.1m/z and clopidogrel- 322 to 154.9m/z) were done in multiple reactions monitoring (MRM) mode and weights were analyzed by using Linear ion trap quadrupole mass spectrometer with turbo spray ion source. The developed method has been successfully used for quantification of drug concentration of sofosbuvir and GS331007 to see the safety of sofosbuvir in patients of HCV infection with renal failures.

Reversed phase UPLC/APCI-MS determination of Vitamin K1 and menaquinone-4 in human plasma: Application to a clinical study

A rapid and sensitive liquid chromatographic-tandem mass spectrometric method was developed and validated to simultaneously quantitate vitamin K1 (PK) and vitamin K2 (MK-4) concentrations in human plasma to evaluate nutritional interventions. Charcoal-stripped human plasma was used for standard and calibration preparation with vitamin E-d6 as the internal standard. Patient plasma samples were extracted using n-hexane and analytes separated using an ACE –PFP C18 column (50 × 2.1 mm, 3 μ) equipped with standard C18 guard. The mobile phase consisted of 0.1 % formic acid in water and 0.1 % formic acid in methanol at a flow rate of 0.5 mL/min. Analyte quantification was achieved by using MS/MS with a positive atmospheric pressure chemical ionization in multiple reaction monitoring (MRM) mode. The lower limit of quantification was 0.01 ng/mL for both PK and MK-4. The assay was linear over the concentration range from 0.01–50 ng/mL for all analytes with a determination coefficient (r2) of 0.998 or better. The intra-and inter-day accuracy and precision were within the acceptable limits per FDA guidance. The validated method was successfully applied to a clinical study for quantification of PK and MK-4 in human plasma to assess nutritional status and dietary interventions in patients with neurodegenerative diseases. Baseline plasma concentrations of PK and MK-4 ranged from 0.23 to 1.81 ng/mL and 0.33–0.57 ng/mL, respectively.