Phase Gradient Elution Research Articles

Simultaneous content determination of notoginsenoside R1, ginsenoside Rg1, Re, Rb1 and dracorhodin in ZJHX rubber paste by double wavelength HPLC

Notoginsenoside R1, ginsenoside Rg1, Re, Rb, and dracorhodin from ZJHX rubber paste were analyzed simultaneously by HPLC, with acetonitrile-water as the mobile phase for gradient elution at a flow rate of 1.0 mL x min(-1). The column temperature was 35 degrees C and the sample size was 10 microL. The detection wavelength was set at 203 nm for ginsenoside and 440 nm for dracorhodin, respectively. The results showed that all of notoginsenoside R1, ginsenoside Rg1, Re, Rb1 and dracorhodin could be were separated well by baseline, with the linear ranges of 0.251-5.020 microg (R2 = 0.999 8), 0.520-10.400 microg (R2 = 0.999 9), 0.251-5.010 microg (R2 = 0.999 7), 0.505-10.100 microg (R2 = 0.999 8) and 0.160-3.270 microg (R2 = 0.999 9), respectively. Each component showed a good linear relationship, with the average recoveries ranging from 99.39% to 100.5%. The established method was so simple, accurate and highly reproducible that it could be used for quality control of ZJHX rubber paste.

Simultaneous determination of paeoniflorin, ferulic acid, prim-O-glucosylcimifugin and 4’-O-beta-glucopyranosyl-5-O-methylvisamminol in Zhengtian pills by HPLC

To simultaneously determine paeoniflorin, ferulic acid, prim-O-glucosylcimifugin and 4'-O-beta-glucopyranosyl-5-O-methylvisamminol in Zhengtian pills. In the test, Insertil ODS-C18 column (4.6 mm x 250 mm, 5 microm) was adopted, with acetonitrile-0.05% phosphoric acid solution as the mobile phase for gradient elution. The flow rate was 1.0 mL x min(-1), the column temperature was 30 degrees C and the detection wavelength was 230 nm. According to the results of the test, paeoniflorin, ferulic acid, prim-O-glucosylcimifugin and 4'-O-beta-glucopyranosyl-5-O-methylvisamminol showed good linear relations between peak areas and sample sizes in 11.37-170.5, 2.188-32.82, 2.896-43.44, and 3.000-45.00 mg x L(-1) (r = 0.999 9, 0.999 9, 1.000 0, 1.000 0), respectively. The average recoveries (n = 6) were 102.3% (RSD 1.2%), 99.71% (RSD 1.9%), 101.2% (RSD 1.2%), and 99.40% (RSD 2.4%), respectively. The above four components were determined in five batches of samples by using the established method, with satisfactory results. The method was so simple, accurate and highly reproducible that it could be used for quality control of the four components in Zhengtian pills.

Study on quality evaluation of Cimicifugae Rhizoma from different producing areas by HPLC fingerprint

To establish a fingerprint for Cimicifugae Rhizoma from different producing areas. Column kromasil (4.6 mm x 250 mm, 5 microm) was employed with acetonitrile-0.1% formic acid solution as the mobile phase for gradient elution. The flow rate was 1.0 mL x min(-1), the detection wavelength was 254 nm. Twenty chromatographic peaks were extracted as the common peaks of fingerprint, and 21 batches of samples were compared and classified with such methods as similarity evaluation, cluster analysis and principle component analysis. The results showed 12 common peaks and three categories of samples. The method was so highly reproducible, simple and reliable that it could provide basis for quality control and evaluation of Cimicifugae Rhizoma from different producing areas.

Analysis on chemical components from water extract of Paeoniae Radix Alba by high performance liquid chromatography-electrospray ionization- quadrupole-time of flight-mass spectrometry

This experiment was performed to establish a qualitative analysis on chemical composition in water extract of Paeoniae Radix Alba by HPLC-ESI-Q-TOF-MS. The analysis was conducted on a C18 (Hanbon Lichrospher, 4.6 mm x 250 mm, 5 microm) column with methanol-0.1% formic acid as the mobile phase for gradient elution; ESI ion source was used for mass spectra, and data were collected in both positive and negative modes. The results showed that eleven compounds from water extract of Paeoniae Radix Alba had been identified by analyzing positive and negative ion mass data including element composition and by comparing with data from literatures. Since efficient separation of HPLC and the high sensitive detection of MS was used, this experiment, it will provide evidences for elucidation of the effective substance in the water extract of Paeoniae Radix Alba.

SIMULTANEOUS DETERMINATION OF DOXORUBICIN AND IRINOTECAN IN CONJUNCTION WITH THEIR MAJOR METABOLITES BY ULTRA HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

An accurate UPLC method was developed and validated for the simultaneous determination of irinotecan and doxorubicin extracted from murine plasma, along with their major metabolites SN-38 and doxorubicinol, respectively. The parent drugs and metabolites were extracted from plasma by protein precipitation method. Camptothecin was used as the internal standard. Chromatographic separation was performed using an ACQUITY UPLC BEH C18 column (150 × 2.1 mm, particle size of 1.7 µm) at 40°C by means of gradient elution. The mobile phases for gradient elution were consisted of 0.1 % v/v formic acid in either acetonitrile or water. The flow rate was 0.5 mL/min, and the total run time was 7 min. The linear quantitation ranges for parent drugs (irinotecan and doxorubicin) and for metabolites (doxorubicinol and SN-38) were 250–10000 ng/mL and 125–5000 ng/mL, respectively (r2 > 0.99). The lower limit of quantification (LLOQ) for irinotecan and doxorubicin was 250 ng/mL, and the LLOQ for SN-38 and doxorubicinol was 125 ng/mL. The intra- and inter-day relative standard deviation was <10 %, and accuracy was >90%.

Simultaneous determination of eleven major components in Fugan Fang using high-performance liquid chromatography coupled with mass spectrometry

Fugan fang (FGF) is a traditional Chinese medicine (TCM) prescription that has been used in treating hepatic illnesses for many years. In this study, an analytical method is developed for the quantitative analysis of the major components of FGF. This method is based on high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS) on a reverse-phase C18 column. Results show that 0.01% acetic acid and acetonitrile is the optimum mobile phase in gradient elution. All compounds showed good linearity (r(2) ≥ 0.9948). Recoveries measured at three concentration levels varied from 83.5 to 104.8%. The method was validated with respect to precision, repeatability and accuracy and was successfully applied in the quantification of the 11 components of FGF products. The validated HPLC-MS method provides a basis for assessing the quality of TCM prescriptions containing many bioactive components.

HPLC-UV METHOD FOR QUANTIFICATION OF SCHIZANDROL A IN RAT PLASMA: VALIDATION AND APPLICATION TO A PHARMACOKINETIC STUDY

Schisandrol A is the most abundant and active component (dibenzocyclooctadiene lignan) isolated from seeds of Schizandra shinensis. it possesses hepatoprotective, antiviral, antioxidant, cytotoxic, and cancer chemopreventive activities (1). The purpose of the present work was to develop and validate a simple and reliable method for the determination of schizan drol A in rat plasma and to use it for the estimation of the pharmacokinetic parameters of schizandrol A. A simple, specific and sensitive RP-h Pl C method with UV detection for the determination of schizandrol A in plasma was developed and validated. from a variety of compounds and solvents tested, abietic acid was selected as the internal standard and acetonitril was found to be the best solvent for extracting schizan drol A from plasma. hPl C analysis of the extracts was performed on a C 18 column (4.6 mm ×150 mm, 5 µm particle size) equipped witch a guard pre-column (same sorbent 2.0 mm). The mobile phase for gradi ent elution consisted of two solvent systems: solvent A 0.03 % (v/v) water solution of trifluoroacetic acid; solvent b methanol. The UV detection was at 250 nm. The elution time for schizandrol A and abietic acid was approximately 7.3 and 25.9 min, respectively. The calibration curve of schizandrol A was linear (r>0.99) over the range 0.10–10 µg/ml in rat plasma. Recovery from plasma was 84–98 %. The limit of detection (l Od) and limit of quantification ( l Oq) for schizandrol A were 0.03 and 0.10 µg/m, respectively. The validated method was successfully applied to the pharmacoki netic study of schizandrol A in rat plasma after single oral administration of Schizandra oil extract (dose of schizandrol A 5 mg/kg). The basic pharmacokinetic parameters of schizandrol A in rats were determined by noncompartmental analysis. The mean AUC 0-t and C max were 3.26 h∙µg/ml and 0.56 µg/ml The peak plas ma levels was achieved to 1.0 hour and the mean elimi nation half life was 3.06 hours. Reference: 1. Min h. y. et al. (2008) bioorg. Med. Chem. lett. 18: 523–526.

Investigation of porous graphitic carbon for triterpenoids and natural resinous materials analysis by high performance liquid chromatography hyphenated to mass spectrometry

Natural resinous materials are mainly composed of pentacyclic triterpenes which exhibit a large number of interesting medicinal activities. However, the presence of numerous isomers within the active substances makes their screening by HPLC very challenging. Porous graphitic carbon was investigated as stationary phase to achieve triterpenes isomers separation. The influence of various parameters (temperature, formic acid concentration and mobile phase composition) on the retention was considered. A usual decrease of the retention of triterpenes was observed with the increase of the temperature. Therefore, separation in resinous materials was performed at 25°C. Acetonitrile–isopropanol mixture was chosen as mobile phase in gradient elution and leads to the best compromise between efficiency and high resolution. The lack of chromophore groups in the pentacyclic triterpenes structures required the use of mass spectrometry detection. Moreover, atmospheric pressure photo-ionisation mass spectrometry prevents compounds fragmentation which was helpful for spectra interpretation and compounds identification.

Simultaneous Quantification of Vitamin D-2, Vitamin D-3, and their 25- Hydroxy Metabolites in Human Plasma by High Performance Liquid Chromatography

A simple and reliable High Performance Liquid Chromatography (HPLC) method for simultaneous determination of vitamin D-2 (VD-2), vitamin D-3 (VD-3), 25-hydroxyvitamin D-2 [25 (OH) VD-2], and 25-hydroxyvitamin D-3 [25(OH) VD-3] in human plasma was developed and validated. Plasma samples were deproteinized with a mixture of methanol and 2-proponol, and extracted with hexane. After evaporation, the residue was dissolved in methanol:water (9.6:0.4, v/v), centrifuged, and then clear solution was injected onto Zorbax C18 column. The mobile phase (gradient elution mode) consists of methanol, acetonitrile, and water (pH = 3.0); the eluents were monitored by photodiode array detector (wavelength set at 265 nm). The relationship between the concentration of VD-2, VD-3, 25(OH) VD-2, 25(OH) VD-3 in plasma and their peak area ratio to the IS was linear over the range of 5 - 100 ng/mL. The coefficients of variation for inter-day and intra-day assay were all ≤ 9.7% and biases ≤ 13.1%. Mean extraction recoveries of VD-2, VD-3, 25(OH) VD-2, and 25(OH) VD-3 from plasma were all over 80%. The method was applied for the determination of vitamin D levels in plasma obtained from healthy subjects. Further, it was used to assess the stability of VD-2, VD-3, 25(OH) VD-2, and 25(OH) VD-3 in plasma under various conditions encountered in the clinical laboratory.

Determination of serotonin and its precursors in chocolate samples by capillary liquid chromatography with mass spectrometry detection

A method for the analysis of serotonin (5-HT) and its precursors, 5-hydroxytryptophan (5-HTP) and l-tryptophan (TP) in chocolate samples by capillary liquid chromatography–mass spectrometry (cLC–MS) has been developed. Optimum chromatographic conditions were established by using a personalized multifactorial experimental design. Finally the cLC separation was achieved through a mixture of acetonitrile and 5mM ammonium formate at pH 4 (3:97, v/v) as mobile phase in gradient elution, setting the injection volume at 10μL and using pure water as injection solvent for focusing purposes on the head of the capillary column. For extraction of targets in chocolate samples a new, fast and simple procedure based on the use of acidic extraction medium and sonication was developed. Working in selected ion mode (m/z 177 for 5-HT, m/z 205 for l-tryptophan and m/z 221 for 5-HTP) detection limits were between 0.01 and 0.11μgg−1 and linearity was in the concentration range of 0.5–25μgg−1. Recoveries higher than 76% with RSDs lower than 8% were obtained from spiked samples for all analytes, showing the effectiveness of the proposed method. Serotonin and its precursors were determined in 5 kinds of commonly consumed chocolates with different cocoa contents (70–100%). The highest serotonin content was found in chocolate with a cocoa content of 85% (2.93μgg−1). Regarding l-tryptophan, the highest content of this amino acid (13.27–13.34μgg−1) was found in chocolate samples with the lowest cocoa content (70–85%). 5-Hydroxytryptophan was not detected in any chocolate samples.

Determination of five boswellic acids in Boswellia serrata

To develop an HPLC method for determinating the contents of five boswellic acids in Boswellia serrata. Analysis was performed on a zorbax SB C18 column eluted with acetonitrile-0.1% phosphoric acid in water as mobile phases in gradient elution and the detection wavelengths were 210 nm and 250 nm. The five ingredients were separated well. The content ranges of alpha-boswellic acid, beta-boswellic acid, 3-acetyl-beta-boswellic acid, 11-keto-beta-boswellic acid and 11-keto-beta-acetyl- boswellic acid were 8.68-16.1, 53.5-246.9, 38.4-192.9, 4.48-5.81, 32.7-44.2 mg x g(-1), respectively. The contents of five individual boswellic acids were different in 12 batches of B. serrata samples.

Study on HPLC fingerprint of 11 Taraxacum species in northeast of China

To study the RP-HPLC fingerprints of 11 plants in the genus Taraxacum for their quality control. The fingerprints were determined using an Agilent 1100 series instrument system. Chromatographic analyses were performed on a Kromasil 100-5 C18 (4.6 mm x 250 mm, 5 microm) analytical column,eluted with methanol and water containing 0.5% acetic acid as the mobile phases in gradient elution at the flow rate of 1.0 mL x min(-1). The detection wavelength was 323 nm. The temperature of column was 35 degrees C. Eleven species of Taraxacum in northeast of China were detected respectively. Twenty-five common peaks existed in 11 RP-HPLC fingerprints. By comparing the retention time and the on-line UV spectra, peaks No. 10, No. 12, No. 16 and No. 25 were identified as chlorogenic acid, caffeic acid, p-coumaroy acid and luteolin respectively. The analytical method with good precision and reproducibility can be useful in the quality control of Taraxacum plants.

Development of Analytical Methods for the Estimation of Alpinetin in Seeds of Amomum subulatum Roxb

The seeds of Amomum subulatum Roxb. is a well known drug in Ayurvedic system of medicine [1]. The seeds mainly contain essential oils, flavonoids and carbohydrates [2]. Alpinetin is a flavonoidal constituent that has recently been reported to posses vasorelaxant and anti-HIV effects [3–5]. In spite of valuable medicinal uses of this drug, there are few methods reported on its quality control [6]. Hence in the present investigations simple, accurate and reliable HPLC and HPTLC methods were developed to quantify alpinetin in amomum seeds for its quality control. RP-HPLC was performed on a C18 column with methanol and water as mobile phase in gradient elution at a flow rate of 1 mL min-1. A sharp peak was obtained for alpinetin at a retention time of 5.7±0.02 minutes. Linear regression analysis showed good linear relationship between response and concentration in the range of 14–224µg mL-1; the regression coefficient was 0.9998. A compact, well resolved peak of alpinetin with Rf value 0.48±0.02 was observed in thin layer chromatography using toluene, ethyl acetate and formic acid as solvent. Standard calibration curve revealed a good linear relationship with r2 value of 0.993 between the peak area and concentration in the range of 20–200µg/spot. Both the methods were validated as per the ICH guidelines and the content of alpinetin was determined which was found in the range of 0.0098 to 0.012% w/w. Statistical analysis proved the methods are precise, reproducible, selective and accurate. Acknowledgment: Author(S) are thankful to CCRUM (AYUSH, ministry of health and family welfare, govt. of India) for financial assistance. Refences: [1] Anonymous (1999) The Ayurvedic Pharmacopoeia of India, Govt. of India, First edition, Part I, Vol. II, 158–159. [2] Shukla SH, Mistry HA (2010) Pharma Science Monitor, 1: 90–102. [3] Wang ZT, Lau CW (2001) Journal of Cardiovascular Pharmacology, 37: 596–606. [4] Tewtrakul S, Subhadhirasakul S (2003) HIV-1 protease inhibitory substances, 504–508. [5] Maridass M, Raju GK (2008) Ethnobotanical Leaflets, 12: 954–994. [6] Li Y, Chou G (2010) Zhongguo Zhong Yao Za Zhi, 35: 2091–2094.

Determination of flavones in different origin and parts of Bupleurum smithii var.parvifoliaum by UPLC-PDA

An ultra performance liquid chromatography method has been developed for determination of flavones in different origin and different parts from Bupleurum smithii var. parvifoliaum. The separation was performed at 30 degrees C on an Acquity UPLC BEH C18 column eluted with methanol and 0.1% phosphoric acid solution as mobile phases in gradient elution. The detection wavelength was set at 256 nm and the flow rate was 0.4 mL x min(-1). There flavones of rutin, quercetin and isorhamnetin were separated well, the linear calibration curves were obtained over the ranges of 0.106-1.06 microg for rutin (r = 0.999 8, n = 6), 0.004 87-0.048 7 microg for quercetin (r = 0. 999 7, n = 6), 0.022 0-0.220 microg for isorhamnetin (r = 0.999 8, n = 6). The mean recoveries of the three compounds were 99.3%, 97.8%, 98.9% with RSD of 2.4%, 3.0%, 3.2%. The result showed that the method is convenient and feasible for determination of the flavone content in B. smithii var. parvifoliaum.

Determination of ilexsideⅡ in leaves of Ilex cornuta by HPLC-ELSD

To develop a HPLC-ELSD method for determination of ilexside II in leaves of Ilex cornuta. The separation was performed on a Waters Syemmetry Shield RP18 column (4.6 mm x 250 mm, 5 microm) and methanol-water was used as mobile phase in gradient elution. The flow was 1.0 mL x min(-1), and the temperature was set at 35 degrees C. The calibration curve showed good linearity in the test range (R2 = 0.9997). And the mean recovery was 101.3%, RSD was 2.0% (n=6). The developed HPLC-ELSD method was accurate and reproducible, and can be used for the determination of ilexside II in leaves of Ilex cornuta.

Determination of vaccarin in Vaccariae Semen by HPLC

To establish a HPLC method for the determination of vaccarin in Vaccariae Semen. Analysis was carried out on an Alltima-C18 column (4.6 mm x 250 mm, 5 microm) eluted with methanol -0.3% phosphoric acid as mobile phase in gradient elution. The flow rate was 1.0 mL x min(-1) and detected wavelength was set at 280 nm. The peak areas and injection ammounts of vaccarin showed a good linear relationship in the range of 0.102-1.539 microg, R2 = 0.9997. The average recovery was 100.4%, RSD was 0.81%. The results of the assay of 10 samples showed that the contents of vaccarin varied in the range of 0.46%-0.57%. The method is simple, accurate, reproducible and specific. It can be used for the quality control of Vaccariae Semen.

A HPLC method to evaluate the influence of photostabilizers on cosmetic formulations containing UV-filters and vitamins A and E

This paper reports a simple and reliable HPLC method to evaluate the influence of two currently available photostabilizers on cosmetic formulations containing combined UV-filters and vitamins A and E. Vitamins and UV-filters, widely encountered in products of daily use have to be routinely evaluated since photoinstability can lead to reductions in their efficacy and safety. UV-irradiated formulation samples were submitted to a procedure that included a reliable, precise and specific HPLC method employing a C18 column and detection at 325 and 235 nm. Methanol, isopropanol and water were the mobile phases in gradient elution. The method precision was between 0.28 and 5.07. The photostabilizers studied [diethylhexyl 2,6-naphthalate (DEHN) and benzotriazolyl dodecyl p-cresol (BTDC)], influenced the stability of octyl methoxycinnamate (OMC) associated with vitamins A and E. BTDC was considered the best photostabilizer to vitamins and OMC when the UV-filters were combined with both vitamins A and E.

Study on HPLC-FPS of raw and processed Fructus Polygoni Orientalis

To establish the HPLC fingerprint method of Fructus Polygoni Orientalis before and after processed by choosing taxifolin as reference to compare the changes of chemical composition. HPLC method was emplyed with a gradient elution phase in a flow rate of 1 mL x min(-1) and the detection wavelength of 270 nm. Twenty marker peaks were marked out in the raw samples and 33 marker peaks in the processed product. Methodology met was consistent with the requirement, similarity was exceeded 0.9. This method is stationary, precise and feasible, which provide references of quality control for Fructus Polygoni Orientalis.

Interspecific Difference Assay of UDP-glucuronosyltransferase 1A9 Activities in Liver Microsomes by Ultra-performance Liquid Chromatography-tandem Mass Spectrometry

Aim To establish a UPLC-ESI-MS method for the assay of UDP-glucuronosyltransferases(UGT) 1A9 activities in human and animal liver microsomes, to investigate the interspecific differences of UGT1A9. Methods The probe substrate of propofol ( m/ z 177.3), II phase metabolite of propofol glucuronide (PG) ( m/ z 377.3, [M + Na] +), and internal standard (IS) of 3-acetamidophenol ( m/ z 152.2, [M + H] +) were quantified by selective ion monitor (SIM), using electrospray ionization-MS (ESI-MS), acquity UPLC BEH C 18 column (50 mm × 2.1 mm, 1.7 μm) and a mixture of acetonitrile(B)-0.5% acetic acid(A) as flow phase in gradient elution at flow rate of 0.3 mL·min −1. Result The linearity was within 0.2 to 12.8 μg·mL −1 for PG. The intra- and inter-day precisions were less than 3% and the accuracies ranged from −3.46% to 3.03%. The recoveries of PG from human liver microsomes (HLMs) were 96.5%-98.6%. The method was successfully used to determine the kinetics of propofol glucuronidation in human and different animals liver microsomes. The results indicated that the kinetics of propofol O-glucuronidation in liver micro-somes from human, mouse, rabbit and sheep were fitted to the substrate inhibition equation, whereas the kinetics in rat liver micro-somes was fitted to the two-enzyme Michaelis–Menten equation. The intrinsic clearance ( CL int ) of propofol glucuronidation in different liver microsomes was in the order of mice > rats ( CL int1 + CL int2 )>sheep>rabbits>human. Conclusion The method is rapid, sensitive and selective for assay of UGT1A9 activities in liver microsomes. The results indicated that the kinetics of propofol O-glucuronidation in rat liver microsomes is different from that in HLMs, and the CL int value for propofol O-glucuronidation in liver microsomes from mice, sheep, and rabbit are remarkably higher than that in HLMs. In animal experiments for drug development, these interspecific differences should be taken into consideration.

Bile acid analysis in biological fluids: a novel approach

In contrast to current methods of bile acid analysis that require the separation of bile acids into different groups prior to their analysis, the HPLC method using a reverse phase column and gradient elution that we developed permits the separation and detection of nonconjugated, glycine-conjugated, and esterified bile acids as their fluorescent dimethoxycoumarin esters. The mild conditions for ester formation make possible the identification of allylic bile acids characteristic of metabolic errors in bile acid synthesis. Quantification is obtained using 7 alpha,12 alpha-dihydroxy-5 beta-cholanoic acid as an internal standard. In addition to identification based on retention time, peak-shift strategy is used by treatment of aliquots with cholyglycine hydrolase and/or solvolysis. Loss of the parent peak and appearance of the derivative provide further assurance of the identity of each bile acid in biologic fluids that contain other organic acids.