Reversed-phase C18 Column Research Articles

COMPOSITION AND QUANTIFICATION OF ANTOCIANS IN HEALTHY-DIET POTATO (Solanum tuberosum L.) VARIETIES FOR GROWING AND SELECTION IN THE RUSSIAN FAR EAST

The potato (Solanum tuberosum L.) has been the focus of dietary research in recent decades due to its ability to accumulate phenolic substances (anthocyanins) in tubers. In Russia, such varieties have been created for a number of regions, but in the Far East, the program to increase the content of anthocyanins in potato tubers has recently begun. In the presented study using liquid chromatography and second-order mass spectrometry methods, we profiled anthocyanins and quantified their accumulation for potato varieties of different origin (Russia, Ukraine, Belarus, Kazakhstan, Germany, the Netherlands), which had not previously been characterized by this feature. For the first time, four dietary Russian varieties with an increased content of anthocyanins have been identified. This work aimed to determine the profiles of anthocyanins and their content in tubers in the conditions of the Russian Far East. We used 22 varieties selected for early maturity, productivity, low starch content and different color of tubers and flowers. The anthocyanin profile was double-identified, by the retention time on a C18 reverse phase column with detection of absorbance at wavelength range 400-700 nm and ESI-MS/MS in positive ionization mode. Anthocyanins were quantified spectrophotometrically. The anthocyanins identified in the tubers were delphinidin, petunidin, malvidin, cyanidin, and pelargonidin. Delphinidin and cyanidin were found in mono- and diglycosylated forms. It was revealed that petunidin-3-glucoside is the most common anthocyanin which is present in almost all of the studied varieties. In tubers, depending on varietal specificity, there were from 1 to 5 anthocyanins of those found. Varieties with yellow skin and pink spots on the yellow tuber skin were characterized by a low content of anthocyanins. The pink and dark pink color of the skin positively correlates with the content of pelargonidin-3-glucoside, while petunidin-3-glucoside and cyanidin-3-glucoside give a purple and blue-violet color to the skin of the tuber. The anthocyanin level was the highest in Phioletovii variety (310 mg/kg cyanidin-3-glucoside, 50 mg/kg malvidin, 30 mg/kg delphinidin), Vasilyok variety (150 mg/kg petunidin), and in Mayak (95 mg/kg pelargonidin) and Kuznechanka (78 mg/kg pelargonidin) varieties. In the Phioletovii variety, the cyanidin-3-glucoside prevailed. We recommend to involve varieties with pink, dark pink, purple and blue-purple tuber skin in selection for an increased content of anthocyanins.

СОСТАВ И СОДЕРЖАНИЕ АНТОЦИАНОВ В ДИЕТИЧЕСКИХ СОРТАХ КАРТОФЕЛЯ (Solanum tuberosum L.), ПЕРСПЕКТИВНЫХ ДЛЯ ВЫРАЩИВАНИЯ И СЕЛЕКЦИИ В УСЛОВИЯХ ДАЛЬНЕГО ВОСТОКА РОССИИ

The potato (Solanum tuberosum L.) has been the focus of dietary research in recent decades due to its ability to accumulate phenolic substances (anthocyanins) in tubers. In Russia, such varieties have been created for a number of regions, but in the Far East, the program to increase the content of anthocyanins in potato tubers has recently begun. In the presented study using liquid chromatography and second-order mass spectrometry methods, we profiled anthocyanins and quantified their accumulation for potato varieties of different origin (Russia, Ukraine, Belarus, Kazakhstan, Germany, the Netherlands), which had not previously been characterized by this feature. For the first time, four dietary Russian varieties with an increased content of anthocyanins have been identified. This work aimed to determine the profiles of anthocyanins and their content in tubers in the conditions of the Russian Far East. We used 22 varieties selected for early maturity, productivity, low starch content and different color of tubers and flowers. The anthocyanin profile was double-identified, by the retention time on a C18 reverse phase column with detection of absorbance at wavelength range 400-700 nm and ESI-MS/MS in positive ionization mode. Anthocyanins were quantified spectrophotometrically. The anthocyanins identified in the tubers were delphinidin, petunidin, malvidin, cyanidin, and pelargonidin. Delphinidin and cyanidin were found in mono- and diglycosylated forms. It was revealed that petunidin-3-glucoside is the most common anthocyanin which is present in almost all of the studied varieties. In tubers, depending on varietal specificity, there were from 1 to 5 anthocyanins of those found. Varieties with yellow skin and pink spots on the yellow tuber skin were characterized by a low content of anthocyanins. The pink and dark pink color of the skin positively correlates with the content of pelargonidin-3-glucoside, while petunidin-3-glucoside and cyanidin-3-glucoside give a purple and blue-violet color to the skin of the tuber. The anthocyanin level was the highest in Phioletovii variety (310 mg/kg cyanidin-3-glucoside, 50 mg/kg malvidin, 30 mg/kg delphinidin), Vasilyok variety (150Â mg/kg petunidin), and in Mayak (95 mg/kg pelargonidin) and Kuznechanka (78 mg/kg pelargonidin) varieties. In the Phioletovii variety, the cyanidin-3-glucoside prevailed. We recommend to involve varieties with pink, dark pink, purple and blue-purple tuber skin in selection for an increased content of anthocyanins.

Development of a HPLC Fluorescence Method for Determining Efficacy of Milk Pasteurization

Alkaline phosphatase (ALP) is an active enzyme present in the raw milk. Due to its unique heat-sensitive properties, it is employed as an indicator to evaluate efficiency of the pasteurization treatment. In the present study, a method comprising of HPLC with fluorescence detector was developed for the quantification of ALP enzyme activity in the pasteurized milk. The method is based on the detection and quantification of 4-methylumbelliferone (4MU) liberated by the action of phosphatase enzyme (in the milk) on the substrate 4-methylumbelliferyl phosphate (4MUP). Extraction and purification of 4MU was done by clarification, centrifugation, and solid phase extraction processes. The target compound separation was achieved with the help of reversed-phase C18 column using gradient elution consisting of a binary mobile phase: HPLC grade water and methanol. The fluorescence detection was accomplished with the excitation and emission wavelengths being 365 and 460 nm, respectively. The method validation experiments clearly showed high specificity, good linearity (r2 > 0.9901), low limits of detection (0.349 mg/L) and quantification (LOQ, 0.432 mg/L), accuracy (> 100%), and precision (%RSD < 4.7652). Comparative evaluation against Lovibond comparator method revealed that the HPLC method has an advantage of detecting very low levels of ALP activity and raw milk contamination. This is the first report of detecting such low levels of ALP in the milk by HPLC-fluorescence detector technique. This new chromatographic method can be considered as a substitute to the existing standard methods that employ fluorometric technique for detecting ALP activity in the pasteurized milk. Our method could certainly enhance the quality assurance standards currently employed in the dairy industry.

Stability-indicating Reversed Phase-Ultra Performance Liquid Chromatography Method Development and Validation for Simultaneous Determination of Encorafenib and Binimetinib in Formulation

A simple, accurate and precise stability indicating method was developed for the simultaneous estimation of the encorafenib (ECRB) and binimetinib (BMTB) in a dosage form by UPLC. Chromatographic elution was processed through a HSS C18 (100 x 2.1 mm, 1.8m) reverse phase column and the mobile phase composition of 0.01N KH2PO4 buffer (3.5 pH) and acetonitrile in the proportion of 55:45 was processed thru a column at a flow rate of 1.0 ml/min. Temperature of the column oven was kept at 30.0°C and the wavelength maximum of detection system was set to 294 nm. Retention times of ECRB and BMTB were found to be 0.767 min and 1.130 min respectively. Repeatability of the method was determined in the form of %RSD and findings were 0.3 and 0.6 for ECRB and BMTB respectively. The percentage recovery of the method was found to be 99.59% and 99.70% for ECRB and BMTB respectively. LOD, LOQ values obtained from regression equations of ECRB and BMTB were 0.51, 1.55mg/ml and 1.47, 4.44 mg/ml respectively. Regression equation of ECRB was y = 6684.x + 18102 and BMTB was y = 13118x + 2159. Two analytes were subjected for acid, peroxide, photolytic, alkali, neutral and thermal degradation studies and the results shown that the percentage of degradation was found between 0.76% and 6.88%. Retention times and total run time of two drugs were decreased and the developed method was simple and economical. So, the developed method can be adopted in industries as a regular quality control test for the quantification of ECRB and BMTB.

Liquid chromatography/mass spectrometry analytical determination of gabapentin transformation products by heterogeneous photocatalysis and environmental evaluation.

Gabapentin is a drug used to treat epilepsy and peripheral neuropathic pain. It is an analog of gamma-aminobutyric acid, and it is a selective blocker of voltage-gated calcium channels. The drug is excreted unmetabolized; it is stable in the environment and is classified as a persistent mobile organic contaminant. Because wastewater treatment plants (WWTPs) are not completely efficient, some bioactive molecules may be released unaltered into the environment. The aim of this study was to provide information about degradation pathways of gabapentin in water by studying its photoinduced transformation products (TPs) through laboratory simulation experiments. Gabapentin and its TPs were monitored in influent and effluent water samples from WWTPs in Germany and Italy. The laboratory simulation used heterogeneous photodegradation mediated by titanium dioxide (TiO2 ). Chromatographic separation was achieved using a C18 reverse-phase column, and the structural identification of TPs was performed using high-resolution electrospray ionization high-resolution mass spectrometry (ESI-HRMS) and multistage MSn experiments. Several TPs were observed during TiO2 photodegradation. Nine new compounds were detected, and potential structures were assigned by studying the fragmentation pathways of the [M + H]+ ions of these TPs and gabapentin. Gabapentin and some of the newly identified TPs were found in environmental samples from WWTPs. The developed high-performance liquid chromatography/high-resolution mass spectrometry method was used to identify TPs from gabapentin. It was then successfully applied to real environmental samples to monitor the TPs as potential environmental pollutants.

Orthogonal label and label-free dual pretreatment for targeted profiling of neurotransmitters in enteric nervous system

Neurotransmitter (NT) abnormalities in the enteric nervous system have been reported as crucial roles to regulate the intestinal inflammation and gut immune homeostasis. Capturing quantitative changes at the NT metabolome provides an opportunity to develop an understanding of neuroimmune-mediated inflammation. Given the wide diversity of chemical characterizations in the NTs, only partial coverage of the NT metabolome can be simultaneously quantified in a single-run analysis. Herein, we summarized the distribution of functional groups of compound entries in the NT metabolome. Based on this information, an orthogonal dansyl-labeling and label-free dual pretreatment approach was separately designed to target phenol and amine NTs and tertiary amine and choline NTs. By combining the dansyl-labeled and unlabeled NTs within a single vial, a comprehensive and practical approach was optimized for quantifying high coverage of NT metabolome in a single-run analysis on the reversed-phase C18 column. Method validation indicated good linearity with correlation coefficients (R2) > 0.99, intra- and interday accuracy with relative error < ±20%, and precision with relative standard deviations of ≤15%. With this method, we could simultaneously monitor the alterations of cholines, amines, amino acids, tryptophan and phenylalanine biological pathways in dextran sulphate sodium-induced colitis mice. The measured levels of NT metabolome ranged from 0.0007 to 3.540 μg/mg in intestinal contents and 0.013–154.54 μg/mL in serum samples. The NT metabolism was disrupted by colitis, characterized by the changed NT levels in serum and excessive amino acid NTs accumulation in the intestinal contents. We envisage that the orthogonal approach is of great significance for the comprehensive determination of targeted metabolomics. NTs have the potential to be biomarkers for clinical metabolomics.

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP-UPLC METHOD FOR THE QUANTIFICATION OF BALOXAVIR MARBOXIL IN TABLET FORMULATION

Objective: Aim of the present work is to develop a simple, accurate and precise stability-indicating method for the quantification of baloxavir marboxil (BLMX) in tablet dosage form by UPLC.&#x0D; Methods: Chromatographic elution was processed through a HSS C18 (100 x 2.1 mm, 1.8 mm) reverse phase column and the mobile phase composition of buffer 0.1% orthophosphoric acid and acetonitrile in the ratio of 50:50 was processed through a column at a flow rate of 0.3 ml/min. Column oven temperature was maintained at 30 °C and the detection wavelength was processed at 240 nm.&#x0D; Results: Retention time of BLMX was found to be 0.87 min. Repeatability of the method was determined in the form of %RSD and the value was 0.2. The percentage mean recovery of the method was found to be 99.47%. LOD, LOQ values obtained from the regression equation of BLMX were 0.69 and 2.10 mg/ml, respectively. Regression equation and correlation coefficient values of BLMX were y = 16994x+7179.2 and 0.9996. Drug was subjected for acid, peroxide, photolytic, alkali, neutral and thermal degradation studies and the results shown that the percentage of degradation was found between 5.96% and 9.55%.&#x0D; Conclusion: Retention time and total run time of the drug was decreased and the developed method was simple and economical. So, the developed method can be adopted in industries as a regular quality control test for the quantification of BLMX.

Mass spectrometry-based measurements of cyclic adenosine monophosphate in cells, simplified using reversed phase liquid chromatography with a polar characterized stationary phase

3′, 5′ – Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that is involved in many cellular functions and biological processes. In several cell types, cholera toxin will increase the level of cAMP, which mediates toxic effects on cells. In this context, we have developed a fast and simple method based on extraction with 5% trichloroacetic acid (TCA) and quantitation with liquid chromatography-mass tandem spectrometry (LC-MS/MS) for measuring cAMP in cells. A main feature of the LC-MS method was employing a reversed phase C18 column (2.1 mm × 50 mm, 1.6 µm particles) compatible with a 100% aqueous mobile phase, providing retention of the highly polar analyte. Isocratic separations allowed for fast subsequent injections. Negative mode electrospray ionization detection was performed with a triple quadrupole (QqQ)MS. cAMP was extracted from cell samples (~106 cells per well) and spiked with a labelled internal standard, using 200 µL of 5% TCA. The extraction solvent was fully compatible for direct injection onto the reversed phase column. After 10 min incubation, the supernatant was removed, and 10 µL of the supernatant was directly analysed by LC-MS. The method was characterized by the simplicity of the extraction, and the speed (3 min retention time of cAMP), sensitivity (250 pg/mL detection limit), and selectivity (separation from interferences e.g. isomeric compounds) of the LC-MS method, and could be used for quantitation of cAMP in the range 1–500 ng/mL cell extract.

Determination of Aromatic Compounds Metabolites in Human Urine by Solid Phase Extraction-liquid Chromatography-Tandem Mass Spectrometry

To establish the method based on high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with solid phase extraction (SPE) for simultaneous determination of the biological metabolites of aromatic compounds, including N-acetyl-S-phenyl-L-cysteine (SPMA), N-acetyl-S-benzyl-cysteine (SBMA), p-nitrophenol (PNP), methylhippuric acids (MHA), p-Aminophenol (PAP), mandelic acid (MA), phenylglyoxylic acid (PGA) and 1-hydroxypyrene (1-OHP) in urine. After adding 20 μL of β-glucuronidase and 1 mL ammonium acetate buffer solution in 1 mL of urine, the sample was digested in a 37 ℃ incubator for 20 h. After digestion, the enzymatic hydrolysate was purified by PRIME HLB solid phase extraction column. The target compounds were eluted with 4 mL of acetonitrile and blown to dryness with nitrogen, reconstituted with 0.20 mL of methanol. Injected the sample solution into LC-MS/MS system for analysis after filtering with 0.22 μm filter membrane. LC separation was carried out on a reversed-phase C18 column (2.1 mm×150 mm, 3.5 μm); gradient eluting was performed at a flow rate of 0.2 mL/min. The water containing 0.1% formic acid was used as mobile phase A and methanol was used as mobile phase B. The mass spectrometry was performed with multiple reaction monitoring (MRM) mode, using alternating positive and negative ions, and internal standard curves were used for quantification. The eight metabolites showed good linearity within the range of 1-100 ng/mL, with a correlation coefficients greater than 0.995, and the relative precision deviation (RSDs) was 0.050%-9.95%. The method detection limits (MDLs) of the eight target metabolites were 0.041-0.12 ng/mL. The proposed method was used for urine sample analysis and the spiked recoveries were 80.1%-114.0%. The established method is quick, sensitive and accurate; it meets the requirementof the biological monitoring of aromatic compounds for the general population and occupational population.

Enhanced protocol for quantitative N-linked glycomics analysis using Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT)™.

The analysis of N-linked glycans using liquid chromatography and mass spectrometry (LC-MS) presents significant challenges, particularly owing to their hydrophilic nature. To address these difficulties, a variety of derivatization methods have been developed to facilitate improved ionization and detection sensitivity. One such method, the Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT)™ strategy for labeling glycans, has previously been utilized in the analysis of N- and O-linked glycans in biological samples. To assess the maximum sensitivity and separability of the INLIGHT™ preparation and analysis pipeline, several critical steps were investigated. First, recombinant and nonrecombinant sources of PNGase F were compared to assess variations in the released glycans. Second, modifications in the INLIGHT™ derivatization step were evaluated including temperature optimization, solvent composition changes, reaction condition length and tag concentration. Optimization of the modified method resulted in 20-100 times greater peak areas for the detected N-linked glycans in fetuin and horseradish peroxidase compared with the standard method. Furthermore, the identification of low-abundance glycans, such as (Fuc)1(Gal)2(GlcNAc)4(Man)3(NeuAc)1 and (Gal)3(GlcNAc)5(Man)3(NeuAc)3, was possible. Finally, the optimal LC setup for the INLIGHT™ derivatized N-linked glycan analyses was found to be a C18 reverse-phase (RP) column with mobile phases typical of RPLC.

Development of a reversed phase-high performance liquid chromatographic method for the analysis of glucosamine sulphate in dietary supplement tablets

Glucosamine sulphate can be taken orally from the dietary supplement tablets for the treatment of osteoarthritis. In these tablets, the determination of glucosamine sulphate concentration is very important for the identification of fraudulent products and quality-control studies. In this study, a simple, rapid, accurate and sensitive method was developed for the determination of glucosamine sulphate in the dietary supplement tablets by using isocratic reversed-phase high-performance liquid chromatography with diode array detector (RP-HPLC-DAD). The separation process was performed by Prodigy™ C18 reversed-phase column using an acetonitrile:H2O mixture (50:50 (v/v), pH 4.0) as mobile phase. The method developed was optimized and subsequently validated. Method validation was performed to illustrate the reliability of new analytical method through various validation parameters including linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), selectivity and stability. Relative standard deviation (%) values for the intra-day and inter-day precision studies were low. The calibration curve was linear in the glucosamine sulphate concentration range of 0.05-2.5 mg.L-1. In this method, recovery values were obtained in the range between 85.15% and 118.86% and LOD and LOQ values were determined to be 0.014 mg.L-1 and 0.046 mg.L-1, respectively. Furthermore, all results confirmed that the new method is suitable for the determination of glucosamine sulphate in dietary supplement tablets.

Development of a liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for characterizing caffeine, methylliberine, and theacrine pharmacokinetics in humans

Coffea liberica possesses stimulant properties without accumulating the methylxanthine caffeine. The basis for this peculiar observation is that methylurates (e.g., theacrine and methylliberine) have replaced caffeine. The stimulant properties of methylurates, alone and in combination with caffeine, have recently been investigated. However, human pharmacokinetics and LC-MS/MS methods for simultaneous measurement of methylxanthines and methylurates are lacking. To address this deficiency, we conducted a pharmacokinetic study in which subjects (n = 12) were orally administered caffeine (150 mg), methylliberine (Dynamine™, 100 mg), and theacrine (TeaCrine®, 50 mg) followed by blood sampling over 24 h. Liquid-liquid extraction of plasma samples containing purine alkaloids and internal standard (13C-Caffeine) were analyzed using a C18 reversed-phase column and gradient elution (acetonitrile and water, both containing 0.1% formic acid). A Waters Xevo TQ-S tandem mass spectrometer (positive mode) was used to detect caffeine, methylliberine, theacrine, and IS transitions of m/z 195.11 → 138.01, 225.12 → 168.02, 225.12 → 167.95, and 198.1 → 140.07, respectively. The method was validated for precision, accuracy, selectivity, and linearity and was successfully applied to characterize the oral pharmacokinetics of caffeine, methylliberine, and theacrine in human plasma. Successful development and application of LC-MS/MS-based methods such as ours for the simultaneous measurement of methylxanthines and methylurates are essential for the characterization of potential pharmacokinetic and pharmacodynamic interactions.

LC-HRMS of derivatized urinary estrogens and estrogen metabolites in postmenopausal women

In order to undertake an epidemiologic study relating levels of parent estrogens (estrone and estradiol) and estrogen metabolites (EMs) to other breast cancer risk factors, we have optimized methods for EM quantification with ultra high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). A two-step approach was adopted; the first step comprised method development and evaluation of the method performance. The second step consisted of applying this method to quantify estrogens in postmenopausal women and determine if the observed patterns are consistent with the existing literature and prior knowledge of estrogen metabolism. First, 1-methylimidazole-2-sulfonyl chloride (MIS) was used to derivatize endogenous estrogens and estrogen metabolites in urine from study participants. Since C18 reversed phase columns have not been able to separate all the structurally related EMs, we used a C18-pentafluorophenyl (PFP) column. The parent estrogens and EMs were baseline resolved with distinct retention times on this C18-PFP column using a 30 min gradient. This method was used to quantify the parent estrogens and 13 EMs in urine samples collected in an initial pilot study involving males as well as pre- and peri-menopausal females to assess a range of EM levels in urine samples and enable comparison to the previous literature for assay evaluation. Detection limits ranged from 1 − 20 pg/mL depending on the EM. We evaluated matrix effects and interference as well as the intra- and inter-batch reproducibility including hydrolysis, extraction, derivatization and LC-MS analysis using charcoal-stripped human urine as a matrix. Methods were then applied to the measurement of estrogens in urine samples from 169 postmenopausal women enrolled in an epidemiological study to examine relationships between breast cancer risk, the intestinal microbiome, and urinary EMs. The results from our cohort are comparable to previous reports on urinary EMs in postmenopausal women and enabled thorough evaluation of the method.

Simultaneous Determination of Multiple Active Components from Bushen Pills and Application in a Pharmacokinetic Study in Rats.

Bushen Pills (BSPs), as a traditional Chinese medicine (TCM), is widely used in clinic to enrich Yang, nourish Yin, stem essence, and strengthen kidneys. Two chromatographic methods, liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), were applied to analyze the multiple active components of BSPs in dosage form for quality evaluation and in rat plasma for pharmacokinetics study, respectively. Three active constituents of BSPs, including paeoniflorin (PF), berberine hydrochloride (BBR), and schizandrin (SCH), were simultaneously determined by the established LC-MS method with electrospray ionization (ESI) in positive selected ion monitoring (SIM) mode at m/z 503.1, 336.0, and 455.2. The contents of PF, BBR, and SCH were (6.112 ± 0.166) mg/g, (335.1 ± 14.95) μg/g, and (5.867 ± 0.136) μg/g in BSPs. On this basis, PF and BBR were selected as targeted analytes for the pharmacokinetic study of BSPs in rats. Memantine hydrochloride was used as an internal standard (IS), and the plasma samples were processed by liquid-liquid extraction with ethyl acetate. All the analytes were separated on a C18 reversed phase column, eluted with a mobile phase consisting of acetonitrile-formic acid (0.01%) (25 : 75, v/v), and detected by ESI in the selected ion mode with multiple reaction monitoring (MRM). The target fragment ions were m/z 525.3 ⟶ 449.5 for PF, 336.2 ⟶ 320.2 for BBR, and 180.1 ⟶ 163.1 for IS. The linear ranges of PF and BBR were 5–500 ng/mL and 0.1–20 ng/mL with good linearity (r2 > 0.99). No obvious matrix effect was observed, and acceptable accuracy, precision, recovery, and stability were obtained. The proposed method has been successfully applied to the pharmacokinetic study of BSPs in rats after a single dose.

Development of an HPLC Method for the Determination of Meropenem/Vaborbactam in Biological and Aqueous Matrixes.

A HPLC method was developed and validated to analyze meropenem and vaborbactam simultaneously in murine plasma and saline matrixes. A 60-μL volume of extracted sample was injected onto a 5-μm BDS Phenyl-Hypersil C18 reversed-phase column and analyzed with a UV detector set at 298nm for the first 4.9min and switched to 240nm. The mobile phase contained a mixture of methanol and 25-mM sodium phosphate buffer set at a flow rate of 1.0mL/min for the 16min run time. Cefuroxime was used as the internal standard. The standard curves were linear over a range of 0.25-50μg/mL. The precision and accuracy for 0.25μg/mL (LLQ) in plasma for both compounds were<4.8% and>98.9%, respectively. Interday and intraday precision and accuracy for all QC plasma samples for both compounds were<6.2% and>95.7%, respectively. This methodology details a reproducible assay for both compounds using a single extraction with good accuracy and precision.

HPLC/colorimetry combination method for quantitative analyses of sclerotiorin produced by Penicillium sclerotiorum

In this study, quantitative analysis of sclerotiorin from Penicillium sclerotiorum was established by using HPLC/colorimetry combination method at the condition of unavailability of authentic sclerotiorin compound. Mycelium was extracted with methanol, then analyzed by HPLC (a C18 reverse phase column with acetonitrile/water = 65/35, v/v as mobile phase and detected at 370 nm) and simultaneously measured OD370 with spectrophotometers. The limit of quantitation (LOQ) was 0.5 μg/mL. Recovery studies were performed using the lyophilized mycelia, with each spiked with sclerotiorin at 1 and 10 mg, respectively. The recoveries were found in the range of 97-109%. The coefficients of variation were less than 3.8% with this HPLC / OD370 combination method.

Analyzing aristolochic acids in Chinese herbal preparations using LC/MS/MS

This paper introduces a newly developed method of applying liquid chromatography- tandem mass spectrometry (LC/MS/MS) to the analysis of aristolochic acids (AAs) in Chinese herbal (medicinal) preparations. Sensitive and highly accurate readings were obtained for this study using both a photodiode array (PDA) detector and tandem mass spectrometer. The following optimized conditions for LC/MS/MS were set for this study: Separation was accomplished using a reverse phase C18 column (2.1 × 150 mm, 5 μm). The mobile phase comprised 35% acetonitrile and a 65% aqueous solution containing 0.1% formic acid and 0.1% ammonium acetate at a flow rate of 0.3 mL/min with a split ratio of 1:1 into the PDA detector and the tandem mass spectrometer. MS/MS qualitative analysis was performed at 3.0 kV capillary voltage, with a collision energy level for aristolochic acid I (AA-1) of 10 eV and for aristolochic acid II (AA-II) of 12eV. The electrospray ionization source was operated in the positive mode. AA-I [M + NH4]+ ions at m/z 359 and AA-II [M + NH4]+ ions at m/z 329 were selected as precursor ions for daughter ion scanning. The characteristic daughter ion mass spectra for AA-I and AA-II were generated and studied carefully. Quantification of results was done using the multiple reaction monitoring (MRM) method. The [M + NH4]+ and [(M + NH4)- NH3-CO2]+ ions of both AA-I and AA-II were selected as precursor and daughter ions for the MRM analysis. MS/MS detection limits were defined as 2.0 ng/mL of AA-I, and 2.8 ng/mL of AA-II. The linear regression correlation coefficients of the calibration cure were 0.9992 of AA-I within the range of 0.02-16.00 μg/mL and 0.9988 of AA-II within the range of 0.028-22.40 μg/mL. Relative standard deviations of 0.73% and 10.44% for AA-I, and 1.38% and 6.10% for AA-II were determined for the intraday and interday tests. Recoveries for five differing concentrations of AA-I ranged from 99.0% to 106.9% and, for five differing concentrations of AA-II, ranged from 92.0% to 104.5%. Levels of AA-I and AA-II detected in the 12 commercial Chinese medicinal preparation samples ranged from 11.1 to 3376.0 μg/g and from 5.4 to 725.4 μg/g, respectively.

Determination of patulin in apple juice produced in Isparta, Turkey by HPLC with diode array detection

In this study patulin was determined by HPLC on a reversed phase C18 column with UV detection at 276 nm. Patulin was extracted with ethyl acetate and then was cleaned up by extraction with sodium carbonate solution. The mobile phase and the column used in the HPLC determination were selected to separate patulin from the interfering substances in apple juice, especially 5-hydroxymethylfurfural (HMF). The linear regression coefficient of the standard curve (y = 327x - 292) for patulin at concentrations ranging from 15 to 200 μg/L was 0.9998 (N = 6). The results were corrected for the recovery percentage. The method is rapid and convenient for the determination of patutin in apple juice.

Determination of adenosine, cordycepin and ergosterol contents in cultivated Antrodia camphorata by HPLC method

The concentrations of adenosine and cordycepin, 3'-deoxyadenosine in the hot water extract and ergosterol in the ethanol extract of a cultivated Antrodia camphorata were measured by high performance liquid chromatography (HPLC) method. The procedure were carried out on a reversed-phase C18 column by linear gradient elution, using four eluents as mobile phase and by isocratic elution, using 100% methanol as mobile phase, respectively. The analysis was completed within 20 min. The application of this method for quantitation of adenosine and cordycepin in 5-L and 100-L fermentation from two A.camphorata strains BCRC 35396 and BCRC 35398 were described. Comparison of adenosine, cordycepin and ergosterol contents were made with different treatment of 700-L fermentation broth.

Determination of ganoderic acids in triterpenoid constituents of Ganoderma tsugae

Ganoderma product has the highest sale volume in the health food market in Taiwan. The most widely-used raw material is the species of G. tsuage. Nine triterpenoids were isolated from the fruiting body of G. tsugae and their structures were identified, using 1H NMR and FT-IR, as ganoderic acid A (1), B (2), C (3), D (4), E (5), C5 (6), C6 (7), G (8) and ganoderenic acid D (9). A reverse-phase HPLC method was also established for the determination of these nine triterpenoids. Among these compounds, ganoderic acid E, C5, C6, G and ganoderenic acid D were analyzed for the first time by reverse-phase HPLC. The separation was carried out on a C18 reverse-phase column with a gradient elution of acetonitrile and 2% acetic acid (1/4 and 1/2, v/v) as mobile phase at a flow-rate of 0.8 mL/min and detection at 252 nm. Regression equation revealed the linear relationship (correlation coefficient: 0.9990-0.9999) between the peak-area ratios and concentrations of ganoderic acids. The recoveries of nine ganoderic acids were between 96.85-105.09%. The relative standard deviations of nine ganoderic acids range from 0.8-4.8% (intraday) and 0.7-5.1% (interday). The total contents of the nine ganoderic acids in eight Ganoderma samples were between 0.28-2.20%.