高效液相色谱-串联质谱法测定葡萄中的吡效隆和赤霉素

采用高效液相色谱-串联质谱法（HPLC-MS/MS）建立了葡萄果实中吡效隆（CPPU）和赤霉素（GA3）两种植物生长调节剂残留量的定性定量分析方法。样品用0.5%甲酸水溶液和乙腈（体积比4:1）混合溶剂提取,经Strata-X固相萃取净化,使用Agilent SB-C18 色谱柱（50mm×2.1mm,1.8μm）,以0.5%甲酸水溶液-乙腈为流动相,流量0.3mL/min,采用电喷雾离子化源（ESI）多反应监测（MRM）模式下HPLC-MS/MS检测。在最佳检测条件下,得到吡效隆和赤霉素在2.0～100.0μg/L检测范围内线性关系良好,相关系数大于0.999;检出限(LOD)分别为0.3μg/L、0.5μg/L,定量限(LOQ)分别为1.0μg/L、2.0μg/L,回收率和精密度均能达到要求,表明该法准确,可用于葡萄中吡效隆和赤霉素残留量测定。     

Simultaneous determination of four lignan compounds in Herpetospermum caudigerum by normal-phase high-performance liquid chromatography

A normal-phase high-performance liquid chromatographic method with diode array UV detection is developed for the simultaneous quantitation of four lignan compounds in Herpetospermum caudigerum. This analysis provides a good resolution and reproducibility. Chromatography is carried out with a mobile phase of N-hexane-dichlormethane-methanol (42.5:42.5:5, v/v) at a flow rate of 1.0 mL/min. UV detection is performed at 280 nm. The calibration curve for lignans concentration is linear over the range of 2.10 to 42.0 microg/mL, 15.26 to 305.2 microg/mL, 6.15 to 123.0 microg/mL, and 6.24 to 124.8 microg/mL, respectively. The limit of quantitation and detection for compounds 1, 2, 3, and 4 is 1.31, 2.74, 2.63, and 2.17 microg/mL and 0.28, 0.25, 0.27, and 0.31 microg/mL, respectively. The validation data show that the assay is sensitive, specific, accurate, and reproducible for the simultaneous quantitation of four compounds. This rapid method is therefore appropriate to quantitate these lignans in Herpetospermum caudigerum.     

Determination of gambogic acid in dog plasma by high-performance liquid chromatography for a pharmacokinetic study

A rapid, simple and sensitive high-performance liquid chromatography method for the quantification of gambogic acid in dog plasma was developed and validated. After acidification with hydrochloric acid, dog plasma was extracted with ethyl acetate and determined by HPLC. The analysis was carried out on a reversed-phase C(18) analytical column. The mobile phase consisted of a mixture of methanol-0.05% phosphoric acid (94:6, v/v), and the column temperature was maintained at 35 degrees C. A constant mobile phase flow rate of 1.0 mL/min was employed throughout the analyses. The ultraviolet detector was set at 360 nm. Chromatographic separation was achieved in less than 10 min and the calibration curve was linear over a concentration range of 0.156-20 microg/mL. The intra-assay and inter-assay variability values were less than 10.0%. The accuracy ranged from 93.0 to 104.2%. The established method has been successfully applied to a pharmacokinetic study of gambogic acid in dogs.     

Determination of foscarnet (trisodium phosphonoformate) in pharmaceutical preparations by high-performance liquid chromatography with ultraviolet detection

An isocratic high-performance liquid chromatographic method with UV detection has been developed and validated for the quantitative determination of foscarnet in isoosmotic sodium chloride aqueous solution. The mobile phase consisted of mixture of methanol:water (30:70 v/v), containing 1 mm tetrahexylammonium hydrogen sulphate at pH 5.80. The analyte was separated on a reversed-phase C(18) column packed with 4 microm spherical particles of octadecylsilane. Hydrochlorothiazide was used as internal standard. UV detection at 232 nm allowed a quantification limit of 50 microg/mL. The assay was linear from 50 to 4000 microg/mL. The coefficient of variation was < or =2.52% for intra-assay precision and < or =3.49% for inter-assay precision. The deviation from the nominal value ranged from -0.57 to 0.47% for the same-day accuracy and from -0.75 to 3.06% for day-to-day accuracy.     

Use of vancomycin silica stationary phase in packed capillary electrochromatography I. Enantiomer separation of basic compounds

Chiral separation of basic compounds was achieved by using 75 or 100 microm ID fused-silica capillaries packed with a vanoomycin-modified diol silica stationary phase. The capillary was firstly packed for about 12 cm with a slurry mixture composed of diolsilica (3:1) then with the vancomycin modified diol-silica (3:1) (23 cm), and finally with diol-silica (3:1) for about 2 cm. Frits were prepared by a heating wire at the two ends of the capillary; the detector window was prepared at 8.5 cm from the end of the capillary where vancomycin was not present. The influence of the mobile phase composition (pH and concentration, organic modifier type and concentration) on the velocity of the electroosmotic flow, chiral resolution and enantioselectivity was studied. Good enantiomeric resolution was achieved for atenolol, oxprenolol, propranolol, and venlafaxine using a mobile phase composition of 100 mM ammonium acetate solution (pH 6)/water/acetonitrile (5:5:90 v/v/v) while for terbutaline a mixture of 5:15:80 v/v/v provided the best separations. The use of methanol instead of acetonitrile caused a general increase of enantiomer resolution of the studied compounds together with a reduction of efficiency and detector response. However, the combination of acetonitrile and methanol in the mobile phase (as, e.g., 10% methanol and 80% acetonitrile) allowed to improve the enantiomer resolution with satisfactory detector response.     

Quantitation of glucosamine from shrimp waste using HPLC

This work presents a high-performance liquid chromatography (HPLC) method for the quantitation of glucosamine in chitin. The method includes an acid hydrolysis of chitin. The chromatographic separation is achieved using a Hypersil ODS 5-microm column (250 x 4.6 mm) at 38 degrees C, with precolumn derivatization with 9-fluorenylmethyl-chloroformate and UV detection (lambda = 264 nm). The mobile phase is a mixture of mobile phase A [30 mM ammonium phosphate (pH 6.5) in 15:85 methanol-water (v/v)], mobile phase B [15:85 methanol-water (v/v)], and mobile phase C [90:10 acetonitrile-water (v/v)], with a flow rate of 1.2 mL/min. The HPLC method proposed showed adequate repeatability (relative standard deviation, 5.8%), accuracy (92.7% recovery), and sensitivity, with a detection limit of 2 microg/mL. The method is successfully applied to the quantitation of glucosamine for the determination of the purity of chitin from shrimp waste.     

反相高效液相色谱法同时测定片剂中的加替沙星与盐酸氨溴索

 A reversed-phase high performance liquid chromatography (HPLC) method was developed, validated, and used for the quantitative determination of gatifloxacin (GA) and ambroxol hydrochloride (AM), from its tablet dosage form. Chromatographic separation was performed on a HiQ Sil C18 column (250 mm×4.6 mm, 5 μm), with a mobile phase comprising of a mixture of 0.01 mol/L potassium dihydrogen orthophosphate buffer and acetonitrile (70∶30, v/v), and pH adjusted to 3 with orthophosphoric acid, at a flow rate of 1 mL/min, with detection at 247 nm. Separation was completed in less than 10 min. As per International Conference on Harmonisation (ICH) guidelines the method was validated for linearity, accuracy, precision, limit of quantitation, limit of detection, and robustness. Linearity of GA was found to be in the range of 10-60 μg/mL and that for AM was found to be 5-30 μg/mL. The correlation coefficients were 0.9996 and 0.9993 for GA and AM respectively. The results of the tablet analysis (n=5) were found to be 99.94% with ±0.25% standard deviation (SD) and 99.98% with±0.36% SD for GA and AM respectively. Percent recovery of GA was found to be 99.92%-100.02% and that of AM was 99.86%-100.16%. The assay experiment shows that the method is free from interference of excipients. This demonstrates that the developed HPLC method is simple, linear, precise, and accurate, and can be conveniently adopted for the routine quality control analysis of the tablet.     

Reversed-phase capillary electrochromatography for the simultaneous determination of acetylsalicylic acid, paracetamol, and caffeine in analgesic tablets

The separation and simultaneous determination of caffeine, paracetamol, and acetylsalicylic acid in two analgesic tablet formulations was investigated by capillary electrochromatography (CEC). The effect of mobile phase composition on the separation and peak efficiency of the three analytes was studied and evaluated; in particular, the influence of buffer type, buffer pH, and acetonitrile content of the mobile phase was investigated. The analyses were carried out under optimized separation conditions, using a full-packed silica capillary (75 microm ID; 30.0 cm and 21.5 cm total and effective lengths, respectively) with a 5 microm C8 stationary phase. A mixture of 25 mM ammonium formate at pH 3.0 and acetonitrile (30:70 v/v) was used as the mobile phase. UV detection was at 210 nm. Good linearity was found in the range of 50-200, 20-160, and 4-20 microg/mL for acetylsalicylic acid (r2=0.9988), paracetamol (r2=0.9990) and caffeine (r2=0.9990), respectively. Intermediate precision (RSD interday) as low as 0.1-0.8% was found for retention times, while the RSD values for the peak area ratios (Aanalyte/AIS) were in the range of 1.9-2.9%. The optimized CEC method was applied to the analysis of the studied compounds present in commercial tablets.     

Development of an ion-pair reversed-phase HPLC method with indirect UV detection for determination of phosphates and phosphites as impurities in sodium risedronate

A method based on RP-HPLC with indirect UV detection was developed for the determination of phosphates and phosphites as impurities in sodium risedronate. RP separation of the phosphates and phosphites was achieved by adding tetrabutylammonium hydroxide as an ion-pairing agent in the mobile phase. Potassium hydrogen phthalate was added to the mobile phase as an ionic chromophore in order to obtain high background absorption of the mobile phase. Separation was performed on a C18 column using a mixture of pH 8.2 buffer (containing 0.5 mM tetrabutylammonium hydroxide and 1 mM phthalate) and acetonitrile (95 + 5, v/v) as the mobile phase, with indirect UV detection at 248 nm. The validation of the method included determination of specificity/selectivity, linearity, LOD, LOQ, accuracy, precision, and robustness. The LOD was 0.86 microg/mL for phosphates and 0.76 microg/mL for phosphites. The LOQ was 2.60 microg/mL for phosphates and 2.29 microg/mL for phosphites. The developed method is suitable for quantitative determination of phosphates and phosphites as impurities in QC of sodium risedronate.     

Validated HPLC method for determination of carboxylic acid metabolite of clopidogrel in human plasma and its application to a pharmacokinetic study

A new, simple, and reproducible method for determination of carboxylic acid metabolite of clopidogrel in human plasma has been developed. After liquid-liquid extraction in acidic medium with chloroform, samples were quantified on a Nova-pak C(8), 5 microm column using a mixture of 30 mM K(2)HPO(4)-THF-acetonitrile (pH = 3, 79:2:19, v/v/v) as mobile phase with UV detection at 220 nm. The flow rate was set at 0.9 mL/min. Ticlopidine was used as internal standard and the total run time of analysis was about 12 min. The method was linear over the range of 0.2-10 microg/mL of clopidogrel metabolite in plasma (r(2) > 0.999). The within-day and between-day precision values were in the range 1.0-4.8%. The limit of quantification of the method was 0.2 microg/mL. The method was successfully used to study the pharmacokinetics of clopidogrel in healthy volunteers.     

HPLC method for the simultaneous determination of atenolol and chlorthalidone in human breast milk

A high-performance liquid chromatographic method was optimized and validated for the determination of atenolol and chlorthalidone (CT) in human breast milk. The milk samples were extracted and purified using ACN and phosphoric acid for precipitation of proteins followed by removal of ACN and milk fats by extraction with methylene chloride. The samples were applied, after an extraction procedure, to a cyanide column using a mobile phase consisting of ACN/water (35:65 v/v) and buffered at pH 4.0 with flow rate of 1.0 mL/min. Quantitation was achieved with UV detection at 225 nm using guaifenesin as the internal standard. The effectiveness of protein precipitation and clean up procedure were investigated. The method was validated over the range of 0.3-20 microg/mL for atenolol and 0.25-5 microg/mL for CT.     

Enantiomeric separation of chlorophenoxy acid herbicides by nano liquid chromatography-UV detection on a vancomycin-based chiral stationary phase

Enantiomeric separation of mecoprop, dichlorprop, and fenoprop herbicides in their acid form, commonly used to control the growth of broad-leaved weeds, was carried out by nano-liquid chromatography (nano-LC) at a flow rate of 60 nL/min, using a packed capillary column with vancomycin-modified silica particles of 5 microm. The length of chiral stationary phase was 21 cm, while the total and effective lengths were 43 and 33cm, respectively. Inner diameter was 0.075 mm. Separated peaks were detected at 195 nm. Several mixtures of methanol, water, and 500 mM ammonium acetate buffer at different pH's were tested as mobile phase, and experimental parameters such as resolution (Rs), capacity factor (k), efficiency (N/m), and enantioselectivity factor (alpha) were measured under all the test conditions. Baseline enantiomeric separation was obtained for the three studied herbicides with alpha in the range 1.6-1.9, using as the mobile phase aqueous solutions containing 85% methanol, 5% of 500mM ammonium acetate pH4.5 buffer, and 10% water. Experimental results show that the vancomycin stationary phase presents a great enantiorecognition capability towards chlorophenoxy acid herbicides on using nano-LC.     

Use of tert-butylbenzoylated tartardiamide chiral stationary phase for the enantiomeric resolution of acidic compounds by nano-liquid chromatography

Several racemic acidic compounds of pharmaceutical and environmental interest have been separated into their enantiomers by nano-liquid chromatography (nano-LC) employing a tert-butylbenzoylated tartardiamide chiral stationary phase (CHI-TBB). CHI-TBB was packed into a fused silica capillary of 100 microm id and retained by two frits made with a heated wire; detection was on-column at a window (about 0.5 cm) prepared by removing the polyimide layer. The normal phase mode was selected for eluting the studied acidic compounds and therefore n-hexane/2-propanol/acetic acid (89/10/1, v/v/v) was used as mobile phase. Working at a flow rate of 220 nL/min a good resolution was obtained for mecoprop, dichlorprop, diclofop, fenoxaprop (herbicides) and for DF 1738Y, DF 1770Y, DF 2008Y (drugs under evaluation). In order to optimize the chiral resolution we modified the polarity of the mobile phase by adding several polar additives such as ethyl acetate, dichloromethane, tert-butyl methyl ether. Better results were obtained for some herbicides on working with 2-propanol/CH2Cl2/n-hexane/acetic acid (8/4/87/1, v/v/v/v). The influence of the capillary temperature on chiral resolution was studied for two herbicides with different chemical structures, namely mecoprop and haloxyfop in the temperature range between 10 and 40 degrees C and with n-hexane/2-propanol/1% acetic acid (89/10/1, v/v/v) as the mobile phase. Linear correlation of ln k vs 1/T and In alpha vs 1/ T was observed; deltaH degrees values were negative, demonstrating that retention of analytes was an exothermic process. A decrease in resolution was observed with rising temperature, showing that enantioresolution was mainly influenced by selectivity factors.     

High-performance liquid chromatographic determination of glycogen in sea urchin gonads with refractive index detection

A high-performance liquid chromatographic method using refractive index detection for the determination of glycogen in sea urchin (Paracentrotus lividus) gonads is developed. After alkaline digestion with sodium carbonate, samples are adjusted to pH 4.6 with citric acid and incubated with amyloglucosidase to hydrolyze the glycogen. The resulting glucose is determined using a Spherisorb NH2 column as the stationary phase and an acetonitrile-water mixture (80:20, v/v) as the mobile phase. The relative standard deviation (%) was 3.57, the limit of detection was 40.1 microg/mL, and the recovery percentage was 97.2%.     

反相高效液相色谱法测定生物转化体系中的甘草酸

采用高效液相色谱法在Hypersil C18色谱柱(4.6 mm i.d.×250 mm,5 μm)上以甲醇-水-冰醋酸(70∶30∶1, 体积比)为流动相分离测定了甘草酸单铵盐生物(酶)转化体系中的甘草酸,流动相流速为1.0 mL/min,紫外检测波长254 nm。实验结果表明,该方法在进样量为0.2~20 μg时具有良好的线性;样品的加标回收率为98%～103％,相应的相对标准偏差为0.16％～1.58％。方法简便、快速、可靠。     

反相高效液相色谱法分离测定墨旱莲中的鳢肠醛

对墨旱莲中的有效成分鳢肠醛进行了分离和结构鉴定,并对其含量进行了分析。色谱条件:Kromasil C18柱(200 mm×4.6 mm,5 μm),流动相为乙腈-0.1%磷酸水溶液(体积比为65∶35),流速为1.0 mL/min,柱温为30 ℃,检测波长为365 nm,进样量为10 μL。结果表明,鳢肠醛的峰面积与其质量浓度有良好的线性关系(r=0.9993)。方法的加样回收率为96.7%～100.0%。该方法简便、快速、准确,可作为墨旱莲质量控制的一个有效方法。     

High-performance liquid chromatographic method for the determination and pharmacokinetic study of wogonoside in rat serum after oral administration of traditional Chinese medicinal preparation Huang-Lian-Jie-Du decoction

A high-performance liquid chromatographic method for the determination of wogonoside in plasma of rats administrated orally with the traditional Chinese medicinal preparation Huang-Lian-Jie-Du decoction was developed. Sample preparation was carried out by protein precipitation with a mixture of acetonitrile and methanol (1:1, v/v). The extracted sample was separated on a Hypersil C(18) (150 x 5 mm i.d., 5 microm) analytical column by linear gradient elution using 0.05% (v/v) phosphoric acid (containing 5 mm sodium dihydrogen phosphate) and acetonitrile as mobile phase at a flow rate of 1.5 mL/min. The eluate was detected using a UV detector at 276 nm. The assay was linear over the range 0.109-7.0 microg/mL (R(2) = 0.9999, n = 5). Mean recovery was determined as 98.39%. Intra- and inter-day precisions (RSD) were < or =7.59%. The limit of quantitation was 0.109 microg/mL. After validation, the HPLC method developed was applied to investigate the preliminary pharmacokinetics of wogonoside in rat after oral administration of Huang-Lian-Jie-Du decoction.     

高效液相色谱法同时测定化妆品中的7种磺胺及甲硝唑和氯霉素

建立了化妆品中7种磺胺(磺胺醋酰、磺胺吡啶、磺胺甲基嘧啶、磺胺二甲嘧啶、磺胺甲氧嘧啶、磺胺间甲氧嘧啶、磺胺甲基异唑)和甲硝唑及氯霉素的高效液相色谱测定方法。样品经0.1%甲酸水溶液-乙腈(体积比为8∶2)混合液超声提取后进行液相色谱分析。方法的定量检测限为3～80 μg/g,7种磺胺在20～200 μg/mL时,甲硝唑及氯霉素在40～400 μg/mL时方法的线性关系良好(r≥0.9993)。加标回收率为83.8%～105.3%(7种磺胺的添加水平为50 μg/mL和150 μg/mL,甲硝唑及氯霉素的添加水平为100 μg/mL和300 μg/mL),其相对标准偏差均小于5%。     

A solid-phase extraction method for high-performance liquid chromatographic determination of salvianolic acid B in rabbit plasma: application to pharmacokinetic study

A sensitive solid-phase extraction/high-performance liquid chromatographic method with ultraviolet detection was established for the analysis of salvianolic acid B in rabbit plasma. The analyte was separated on a reversed-phase column with trifluoroacetic acid-methanol-acetonitrile (70:10:20, v/v/v) as mobile phase at a flow rate of 1 mL/min, and ultraviolet detection at 315 nm. The calibration curve for salvianolic acid B was linear over the range 35-1400 microg/L with coefficients of correlation >0.999. The inter-day and intra-day precisions of analysis were <15%, and assay accuracy ranged from 95.3 to 109.1%. This method is suitable for determining salvianolic acid B in plasma and thus investigating the pharmacokinetics of salvianolic acid B.     

Analysis of phenolic compounds in extra virgin olive oil by using reversed-phase capillary electrochromatography

In this work, the simultaneous separation of ten phenolic compounds (protocatechuic, p-coumaric, o-coumaric, vanillic, ferulic, caffeic, syringic acids, hydroxytyrosol, tyrosol and oleuropein) in extra virgin olive oils (EVOOs) by isocratic RP CEC is proposed. A CEC method was optimized in order to completely resolve all the analyzed compounds by studying several experimental parameters. The influence of the stationary phase type (C(18) and C(8) modified silica gel), buffer concentration and pH as well as the organic modifier content of the mobile phase on retention factors, selectivity and efficiency were evaluated in details. A capillary column packed with Cogent bidentate C(18) particles for 23 cm and a mobile phase composed by 100 mM ammonium formate buffer pH 3/H(2)O/ACN (5:65:30 v/v/v) allowed the baseline resolution of the compounds under study in less than 35 min setting the applied voltage and temperature at 22 kV and 20 degrees C, respectively. A study, evaluating the intra- and interday precision as well as LOD and LOQ and method linearity was developed in accordance with the analytical procedures for method validation. LODs were in the range of 0.015-2.5 microg/mL, while calibration curves showed a good linearity (r(2) >0.997). The CEC method was applied to the separation and determination of these compounds in EVOO samples after a suitable liquid-liquid extraction procedure. The mean recovery values of the studied compounds ranged between 87 and 99%.