Determination of mangiferin, jateorrhizine, palmatine, berberine, cinnamic acid, and cinnamaldehyde in the traditional Chinese medicinal preparation Zi-Shen pill by high-performance liquid chromatography

High-performance liquid chromatography is employed to determine the contents of six marker components such as mangiferin, jateorrhizine, palmatine, berberine, cinnamic acid, and cinnamaldehyde in the traditional Chinese medicinal preparation Zi-Shen pill. The separation is performed on a C(18) column by stepwise gradient elution with water (0.2%, v/v, triethylamine adjusted to pH 4 with phosphoric acid)-methanol-acetonitrile (0.01 min, 98:0:2; 20 min, 80:5:15; 30 min, 65:13:22; and 55 min, 65:13:22) as the mobile phase at a flow rate of 0.9 mL/min, with UV detection at 280 nm. Six regression equations show good linear relationships between the peak area of each marker and concentration. The recoveries of the markers listed are 95.5%, 98.3%, 96.8%, 99.5%, 101.7%, and 102.1%, respectively. The repeatability and reproducibility (relative standard deviation) of the method are less than 2.5% and 3.3%, respectively.     

Quantitative HPTLC determination of prostaglandin E1

Direct in situ determination of PGE₁ in dioxane/water (1:1) at concentrations between 50 μg/ml and 1 mg/ml is described. HPTLC of PGE₁ was carried out on HPTLC Kieselgel 60 o.F. plates; application (200 nl) was with a Nano-Appllcator and ethyl acetate/formic acid (400:5, v/v) served as mobile phase. The spots were located by dipping the plates into a 3% cupric acetate solution in 15% aqueous phosphoric acid, followed by heating at 120°C for 15 min. The plates were then evaluated directly at λ = 345 nm by chromatogram spectrometry. The coefficient of variation is 3.6%.     

High-performance liquid chromatographic analysis of the anticancer drug oxantrazole in rat whole blood and tissues.

A reversed-phase high-performance liquid chromatographic (HPLC) assay was developed for the antitumor anthrapyrazole analogue, oxantrazole (OX), in rat whole blood and tissues. Blood samples were mixed with equal volumes of a 25% (w/v) aqueous solution of L-ascorbic acid, whereas tissue samples were homogenized with 1.5-3 volumes of an L-ascorbic acid-methanol-water (1:10:1, w/v/v) mixture to prevent oxidative degradation of OX. Samples were then treated with 60% (v/v) perchloric acid (25-30 microliters/ml of stabilized sample) to precipitate proteins, and centrifuged, with the resultant supernatants analyzed on HPLC utilizing a C8 column. The mobile phase for blood and urine samples consisted of 8% (v/v) glacial acetic acid, 13% (v/v) acetonitrile, 79% (v/v) water, 0.16% (w/v) sodium acetate, and 0.05% (w/v) L-ascorbic acid (final pH 2.7), and was pumped at 1.8 ml/min. Tissue samples were eluted at 2 ml/min with a mobile phase consisting of 8% (v/v) glacial acetic acid, 12% (v/v) acetonitrile, 80% (v/v) water, 0.16% (w/v) sodium acetate, and 0.0;5% (w/v) L-ascorbic acid. OX and internal standard were detected at 514 nm and had retention times of 2.3 and 3.1 min, respectively. The limit of quantitation of OX was 25-50 ng/g. Recovery of OX from biological samples ranged from 50 +/- 0.9% in spleen to 102.8 +/- 1.8% in RG-2 glioma. The analytical method was applied to a pharmacokinetic study in rats.     

Liquid chromatographic/electrospray mass spectrometric determination (LC/ESI-MS) of chelerythrine and dihydrochelerythrine in near-critical CO2 extracts from real and spiked plasma samples

Two polar benzo[c]phenanthridine alkaloids, chelerythrine (CHE) and dihydrochelerythrine (DHCHE), were extracted at 35 °C and 10 MPa (15 MPa for real samples) from real and spiked plasma samples with acceptable recoveries (95.1% and 81.0%, respectively) using near-critical CO₂ modified with aqueous (1:1, v/v) methanol. The alkaloids were quantified by a liquid chromatographic/electrospray mass spectrometric (LC/ESI-MS) method on a Zorbax SB-CN column (75 mm × 4.6 mm, 3.5 μm particle size) using methanol (organic phase) and 50 mM ammonium formiate (aqueous phase) as a mobile phase. A linear gradient 0-1 min, isocratic at 60% organic phase (v/v); from 1.0 to 7.0 min, 60-71% organic phase (v/v); from 7.0 to 18.0 min, 71-60% organic phase (v/v) was applied. The limit of detection was 1.22 ng (3.50 pmol) for CHE and 0.95 ng (2.72 pmol) for DHCHE per 1 ml of the sample. The linearity of the calibration curves was satisfactory as indicated by coefficients of determination 0.9979 and 0.9995 for CHE and DHCHE, respectively. Repeatability and intermediate precision (average R.S.D.s) were 1.0-1.5%, accuracy was in the range 99.7-100.3%. Average recovery was 100.1% for both, standard solutions and spiked plasma extracts. Three samples of real rat plasma were extracted and analysed to test the method.     

高效液相色谱法分析环境样品中的苯基胂化合物

二苯氯胂和二苯氰胂是刺激性毒剂,在环境中易于降解,其产物苯胂酸、苯胂氧、二苯胂酸、氧联双二苯胂和三苯胂比较稳定,对环境危害大。建立了同时测定这5种含胂产物的反相高效液相色谱方法,选择了最佳色谱条件,提供了各组分的紫外光谱图。5种化合物的线性范围分别为8~30,5~40,20~4000,120~8000,1~60 mg/L,检测限分别为0.1,0.1,0.2,10,0.1 mg/L。对实际环境样品进行了分析,结果较好。     

Study of tanshinone IIA tissue distribution in rat by liquid chromatography-tandem mass spectrometry method

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for determining tanshinone IIA in rat tissues. After a single step liquid-liquid extraction with diethyl ether, tanshinone IIA and loratadine (internal standard) was subjected to LC/MS/MS analysis using positive electro-spray ionization under selected reaction monitoring mode. Chromatographic separation of tanshinone IIA and loratadine was achieved on a Hypersil BDS C(18) column (i.d. 2.1 x 50 mm, 5 microm) with a mobile phase consisting of methanol-1% formic acid (90:10, v/v) at a flow rate of 300 microL/min. The intra-day and inter-day precision of the method were less than 10.2 and 12.4%, respectively. The intra-day and inter-day accuracies ranged from 99.7 to 109.7%. The lowest limit of quantification for tanshinone IIA was 1 ng/mL. The method was applied to a tanshinone IIA tissue distribution study after an oral dose of 60 mg/kg to rats. Tanshinone IIA tissue concentrations decreased in the order of stomach > small intestine > lung > liver > fat > muscle > kidneys > spleen > heart > plasma > brain > testes. Tanshinone IIA still could be detected in most of the tissues at 20 h post-dosing. These results indicate that the LC/MS/MS method was rapid and sensitive to quantify tanshinone IIA in different rat tissues.     

Simultaneous determination of niflumic acid and its prodrug, talniflumate in human plasma by high performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of niflumic acid and its prodrug, talniflumate, in human plasma. Niflumic acid and talniflumate were eluted isocratically with methanol-water (73:27, v/v, adjusted to pH 3.5 by acetic acid) at a fl ow rate of 1 mL/min. Indomethacin was used as an internal standard. Signals were monitored by an UV detector at 288 nm. Retention times of indomethacin, niflumic acid and talniflumate were 5.9, 7.2 and 13.5 min, respectively. Calibration plots were linear over the range 50-5000 ng/mL for niflumic acid and 100-5000 ng/mL for talniflumate. The limits of quantitation were 50 ng/mL for niflumic acid and 100 ng/mL for talniflumate. The intra- and inter-day relative standard deviations (RSD) of niflumic acid and talniflumate were less than 10% and the accuracies were higher than 90%. This method is rapid, sensitive and reproducible for the determination of niflumic acid and talniflumate in human plasma.     

Application of liquid chromatography to the simultaneous determination of acetylsalicylic acid, caffeine, codeine, paracetamol, pyridoxine, and thiamine in pharmaceutical preparations.

This paper describes a rapid reversed-phase liquid chromatographic method, with UV detection, for the simultaneous determination of acetylsalicylic acid, caffeine, codeine, paracetamol, pyridoxine, and thiamine in pharmaceutical preparations. A reversed-phase C18 Nucleosil column is used. The mobile phase consists of 2 successive eluants: water (5 min) and acetonitrile-water (75 + 25, v/v; 9 min), both adjusted to pH 2.1 with phosphoric acid. Before determination acetylsalicylic acid is completely converted to salicylic acid by alkaline hydrolysis. Salicylic acid, caffeine, paracetamol, pyridoxine, and thiamine are all detected at 285 nm, whereas codeine is detected at 240 nm. Calibration curves were linear for salicylic acid, caffeine, paracetamol, and pyridoxine in the range of 50-500 mg/L, and for codeine and thiamine in the range of 50-1000 mg/L. The method was applied to the analysis of 13 fortified commercial pharmaceutical preparations. Recoveries ranged from 92.6 to 105.5%, with relative standard deviations of 1.1-5.8%.     

Determination of gentiopicroside, mangiferin, palmatine, berberine, baicalin, wogonin and glycyrrhizin in the traditional Chinese medicinal preparation Sann-Joong-Kuey-Jian-Tang by high-performance liquid chromatography

High-performance liquid chromatography was employed to determine the contents of several marker substances such as gentiopicroside, mangiferin, palmatine, berberine, baicalin, wogonin and glycyrrhizin in Sann-Joong-Kuey-Jian-Tang. The separation was performed on a Cosmosil 5C₁₈-AR column by gradient elution with 0.03% (v/v) phosphoric acid-acetonitrile (0 min, 90:10; 10 min, 87:13; 17–27 min, 77:23; 40 min, 62:38; 50 min, 55:45) as the mobile phase at a flow-rate of 1.0 ml/min, with detection at 254 nm. n-Propylparaben was used as the internal standard and seven regression equations revealed linear relationships between the peak-area ratios (marker substances/internal standard) and concentrations. The repeatability and reproducibility (relative standard deviation) of the method were in the ranges 0.02−1.78% and 1.44−4.95%, respectively.     

Simultaneous determination of theophylline,tolbutamide, mephenytoin,debrisoquin, and dapsone in human plasma using high‐speed gradient liquid chromatography/tandem mass spectrometry on a silica‐based monolithic column

Theophylline, tolbutamide, mephenytoin, debrisoquin, and dapsone are marker substrates for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, respectively. A silica‐based monolithic column (Chromolith SpeedROD RP‐18e, 50×4.6 mm) was used to separate these five marker substrates of cytochrome P450 within only 84 s. Linear gradient elution was from acetonitrile‐water‐formic acid (10 : 90 : 1, v/v/v) to acetonitrile‐water‐formic acid (90 : 10 : 1, v/v/v) in 1.4 min. The flow rate was 2.5 mL/min. The retention time was 0.52 min for theophylline, 0.67 min for debrisoquin, 0.78 min for dapsone, 0.96 min for mephenytoin, and 1.13 min for tolbutamide. Detection was by tandem mass spectrometry using a PE Sciex API 3000 mass spectrometer with a Turbo‐Ionspray source in positive mode. A simple protein precipitation method was used. This method was validated over the concentration range of 5–2000 ng/mL based on the sample volume of 0.1 mL.     

A simple and rapid HPLC‐UV method for the determination of retigabine in human plasma

A simple and rapid high‐performance liquid chromatographic method with ultraviolet detection was developed for the quantitative determination of retigabine, known also as ezogabine, in human plasma. The assay uses a simple solid‐phase extraction for sample preparation and direct injection of the extract into the chromatograph. Flupirtine is used as an internal standard. Chromatographic separation is achieved on a C₁₈ Chromolith column (Chromolith Performance, 100 × 4.6 mm i.d.), using as mobile phase water/acetonitrile/methanol (72:18:10 v/v/v) mixed with 0.1% of 85% phosphoric acid. Isocratic elution is conducted at a flow rate of 1.5 mL min⁻¹. The total duration of a chromatographic run is 7 min. Calibration curves are linear over the 25–2000 ng mL⁻¹ concentration range, with a limit of quantitation of 25 ng mL⁻¹. Other performance characteristics include high precision (intra‐ and inter‐day coefficients of variation ≤12.6%) and high accuracy (99.7%–108.7%). The method is suitable for the investigation of concentration–response relationships in patients receiving therapeutic doses of retigabine.     

Development and Validation of HPLC,TLC and Derivative Spectrophotometric Methods for the Analysis of Ezetimibe in the Presence of Alkaline Induced Degradation Products

Reversed phase‐high performance liquid chromatography (RP‐HPLC), thin layer chromatography (TLC) densitometry and first derivative spectrophotometry (1D) techniques are developed and validated as a stability‐indicating assay of ezetimibe in the presence of alkaline induced degradation products. RP‐HPLC method involves an isocratic elution on a Phenomenex Luna 5μ C₁₈ column using acetonitrile: water: glacial acetic acid (50:50:0.1 v/v/v) as a mobile phase at a flow rate of 1.5 mL/min. and a UV detector at 235 nm. TLC densitometric method is based on the difference in Rf‐values between the intact drug and its degradation products on aluminum‐packed silica gel 60 F₂₅₄ TLC plates as stationary phase with isopropanol: ammonia 33% (9:1 v/v) as a developing mobile phase. On the fluorescent plates, the spots were located by fluorescence quenching and the densitometric analysis was carried out at 250 nm. Derivative spectrophotometry, the zero‐crossing method, ezetimibe was determined using first derivative at 261 nm in the presence of its degradation products. Calibration graphs of the three suggested methods are linear in the concentration ranges 1–10 mcg/mL, 0.1–1 mg/mL and 1–16 mcg/mL with a mean percentage accuracy of 99.05 ± 0.54%, 99.46 ± 0.63% and 99.24 ± 0.82% of bulk powder, respectively. The three proposed methods were successfully applied for the determination of ezetimibe in raw material and pharmaceutical dosage form; the results were statistically analyzed and compared with those obtained by the reported method. Validation parameters were determined for linearity, accuracy and precision; selectivity and robustness and were assessed by applying the standard addition technique.     

Development and validation of a liquid chromatography-mass spectrometry method for the determination of 4,5-diazafluoren-9-one

A new liquid chromatography-mass spectrometry (LC-MS) method is developed and validated for the identification and determination of novel 4,5-diazafluoren-9-one compound. The method employs a Waters XTerra RP-18 column (150 mmx4.6 mm, i.d. 5 microm) with a mobile phase comprised of a (50:50, v/v) mixture of deionized water containing 0.2% acetic acid (solvent A) and methanol (solvent B) at a flow rate of 1 mL/min, at 35 degrees C. The detection is performed with photodiode-array (PDA) set at 210-400 nm and single quadropole mass spectrometer with electrospray ionization (ESI) positive ion mode. The chromatographic separation is achieved in less than 3 min. The linearity is established over the concentration range of 0.1-0.5 mg/mL (r2=1.000). The mean RSD values for intra- and inter-day precision studies are <2%. The recovery of 4,5-diazafluoren-9-one ranged between 99.84 and 99.97%. The limits of detection and quantitation are determined to be 0.58 and 0.1 mg/mL, respectively.     

Simultaneous micro-determination of nicotinamide and its major metabolites, N1-methyl-2-pyridone-5-carboxamide and N1-methyl-4-pyridone-3-carboxamide, by high-performance liquid chromatography

A simultaneous micro-determination of nicotinamide and its major metabolites, N1-methyl-2-pyridone-5-carboxamide (2-py) and N1-methyl-4-pyridone-3-carboxamide (4-py) by high-performance liquid chromatography is described. The method employs a 7-ODS-L (250 mm X 4.6 mm I.D., particle size 7 microns) column eluted with 10 mM potassium dihydrogenphosphate-acetonitrile (96:4, v/v; pH adjusted to 3.0 by the addition of concentrated phosphoric acid) at a flow-rate of 1.0 ml/min. The UV detector was set at 260 nm. The detection limits for nicotinamide, 2-py and 4-py were 10 pmol (1.22 ng), 2 pmol (304 pg) and 2 pmol (304 pg), respectively, at a signal-to-noise ratio 5:1. Isonicotinamide was used as an internal standard. The technique was applied to the analysis of rat and human urines. The total analysis time was ca. 15 min.     

Isolation and structural characterization of polymyxin B components

Polymyxin B is a peptide antibiotic complex present as sulphate. The components were separated preparatively on a poly(styrene-divinylbenzene) (PLRP-S), 1000 A, 8 microm, 250 x 12.5 mm I.D. stationary phase maintained at 60 degrees C and using 215 nm detection. Elution was carried out with acetonitrile-sodium sulphate solution (0.7%, m/v; pH adjusted to 2.5 with trifluoroacetic acid)-water (18:50:32, v/v) at a flow-rate of 4.0 ml/min. Seven polymyxin B components were isolated and characterized using 1H and 13C NMR. The molecular masses were confirmed by mass spectrometry. The structures of two components were determined for the first time. Polymyxins B5 and B6 were identified as having the same composition as polymyxin B1 except that the fatty acid moiety was nonanoic acid and 3-hydroxy-6-methyloctanoic acid, respectively.     

制备色谱法分离纯化高良姜黄酮中高良姜素和山柰素

建立了制备型高效液相色谱(Prep-HPLC)分离高良姜黄酮中高良姜素和山柰素的方法。高良姜黄酮经HPD-600树脂吸附洗脱纯化后,采用Prep-HPLC分离高良姜黄酮中高良姜素和山柰素。制备色谱条件:流动相为甲醇-0.6%(v/v)乙酸水溶液(58:42, v/v),柱温为常温,流速为7.0 mL/min,检测波长为360 nm,进样量为700 μ L,上样质量浓度为10.0 g/L。分离的单体由质谱和核磁共振氢谱、碳谱鉴定确证为高良姜素和山柰素,HPLC外标法定量,纯度分别为99.5%和99.7%。该方法分离效果好、高效、低毒,可用于高良姜中高良姜素和山柰素的分离制备。     

Development and validation of RP-HPLC,HPTLC and UV-visible spectrophotometric methods for simultaneous estimation of alprazolam and propranolol hydrochloride in their combined dosage form

Three accurate, sensitive and reproducible methods are described for the quantitative determination of alprazolam (ALP) and propranolol hydrochloride (PNL) in their combined dosage form. The first method involves an RP-HPLC separation on the C₁₈ column using acetonitrile-25 mM ammonium acetate buffer and 0.2% triethylamine (pH of buffer adjusted to 4 with glacial acetic acid) in the ratio of 35: 65 (v/v) as mobile phase. Symmetrical peaks with good separation, ALP at 9.3 min and PNL at 3.5 min, were achieved. Quantification was done with photo diode array detection at 255 nm over the concentration ranges of 0.5–50 and 10–250 μg/mL for ALP and PNL, respectively. The second method is based on the separation of drugs by HPTLC using chloroform-methanol-ammonia 7: 0.8: 0.1 (v/v/v) as mobile phase. Quantification was achieved using UV detection at 248 nm over the concentration range of 100–600 ng/spot and 5–30 μg/spot for ALP and PNL, respectively. The third method involves dual wavelength UV-visible spectrophotometric method. It is based on the determination of PNL at 319.4 nm using its absorptivity value and ALP at 258.2 nm after deduction of absorbance due to PNL. Quantification was achieved over the concentration range of 1–40 and 80–200 μg/mL for ALP and PNL, respectively. All methods were validated according to ICH guidelines and successively applied to marketed pharmaceutical formulation, and the results of all three methods were compared statistically as well. No interference from the tablet excipients was found.     

高效液相色谱-二极管阵列检测-电化学检测联用技术同时测定三精双黄连口服液中的4种化合物

建立了应用高效液相色谱-二极管阵列检测-电化学检测(HPLC-DAD-ECD)联用技术同时测定三精双黄连口服液中的绿原酸、咖啡酸、黄芩甙和木樨草素的方法。以Zorbax SB-C18柱(150 mm×4.6 mm i.d., 5.0 μm)为色谱柱,柱温为30 ℃，流动相为(A)甲醇和(B)甲醇-水-冰醋酸(体积比为50∶50∶1),其梯度洗脱程序为2%A3 min3%A12 min25%A5 min80%A。流速为0.8 mL/min。二极管阵列检测波长为275 nm。电化学单安培检测器的工作电压为0.7 V。在上述条件下实现了绿原酸、咖啡酸、黄芩甙和木樨草素的分离检测。上述4种化合物的回收率为96.6%～99.6%,相对标准偏差(RSD)为2.5%～4.1%,检测限依次为1,0.2,9和7 mg/L。该方法简便、快速,重现性和准确度较好,可作为测定双黄连口服液中绿原酸、咖啡酸、黄芩甙和木樨草素的有效方法。     

Efficient methods for isolating five phytochemicals from Gentiana macrophylla using high‐performance countercurrent chromatography

Efficient high‐performance countercurrent chromatography methods were developed to isolate five typical compounds from the extracts of Gentiana macrophylla. n‐Butanol‐soluble extract of G. macrophylla contained three hydrophilic iridoids, loganic acid ( 1 ), swertiamarin ( 2 ) and gentiopicroside ( 3 ), and a chromene derivative, macrophylloside D ( 4 ) which were successfully isolated by flow rate gradient (1.5 mL/min in 0–60 min, 5.0 mL/min in 60–120 min), and consecutive flow rate gradient HPCCC using n‐butanol/0.1% aqueous trifluoroacetic acid (1:1, v/v, normal phase mode) system. The yields of 1 – 4 were 22, 16, 122, and 6 mg, respectively, with purities over 97% in a flow rate gradient high‐performance countercurrent chromatography, and consecutive flow rate gradient high‐performance countercurrent chromatography gave 1 , 2 , 3 (54, 41, 348 mg, respectively, purities over 97%) and 4 (13 mg, purity at 95%) from 750 mg of sample. The main compound in methylene chloride soluble extract, 2‐methoxyanofinic acid, was successfully separated by n‐hexane/ethyl acetate/methanol/water (4:6:4:6, v/v/v/v, flow‐rate: 4 mL/min, reversed phase mode) condition. The structures of five isolates were elucidated by ¹H, ¹³C NMR and ESI‐Q‐TOF‐MS spectroscopic data which were compared with previously reported values.     

Pretreatment of Biodiesel by Esterification of Palmitic Acid in Brønsted–Lowry Acid Based Deep Eutectic Solvents

Biodiesel was pretreated by the esterification of palmitic acid over Brønsted–Lowry acid-based deep eutectic solvents. The optimal deep eutectic solvent was synthesized from choline chloride and trichloroacetic acid (1:1), and the methanol to deep eutectic solvent ratio was 10% by mass. The reaction was optimized at a methanol/palmitic acid ratio of 50:1 at 80°C for 120 min. Under the optimized conditions, good calibration curves were obtained at phenolic acid concentrations ranging from 10.0 to 500.0 µg/mL. The recoveries ranged from 99.7% to 104.2%, and the interday and intraday relative standard deviations were less than 5%. The conversion rate was 94.6%, suggesting deep eutectic solvents will find applications in the preparation of biodiesel.