RP-HPLC法测定重组人生长激素注射液中间甲酚含量

目的：建立测定重组人生长激素注射液中间甲酚含量的方法。方法：采用RP-HPLC法,色谱柱为ShiseidoC4（250mmx4.6mm,5um）,流动相为0.05MTris-HCL（pH7.5）-正丙醇（71：29）,流速0.5ml/min,检测波长为317nm,柱温为40℃。结果：间甲酚在0.2mg-0.8mg/ml（r=0.9996,n=5）浓度范围内与峰面积呈良好线性关系,最低检出线为0.1ng,平均回收率为100.5%（n=9）。结论：本方法准确,重现性好,可为重组人生长激素注射液质量评价提供较为可靠的分析方法。     

8mol/L脲中rhIL-3含量的测定

为解决8mol／L脉中rhIL－3的定量问题,以卵清蛋白作内标,进行常规SDS－PAGE后,作激光灰度扫描,并计算rhIL－3和卵清蛋白的峰面积,发现两种蛋白峰面积的比值与rhIL－3浓度在0．2－1．0mg／ml间呈良好线性关系。查标准曲线可以计算出8mol／L脲中rhIL－3的含量。重复性测定表明该法具有较好的重现性。     

GC 法测定丙酮酸乙酯注射液中EP 的含量

目的:建立测定丙酮酸乙酯的含量测定方法。方法:采用气相色谱法,以环戊酮为内标物。色谱柱为VARIAN CP7502(25 m×0.25 mm×0.25μm),柱温115℃,进样口温度210℃,FID检测器温度210℃,氮气(载气)流量为30 ml.min-1;氢气(燃气)流量为40 ml.min-1;空气(助燃气)流量为400 ml.min-1,分流比1:100。结果:EP进样浓度在0.50035 mg.ml-1～9.0063 mg.ml-1范围内与峰面积积分呈良好的线性关系(r2=0.9996),平均加样回收率为99.76%,RSD为0.46%。结论:本方法简便、快速、准确、重复性好,可用于丙酮酸乙酯注射液的质量控制。     

魔芋中神经酰胺类物质的HPLC-ELSD分析及其含量测定

建立高效液相色谱-蒸发光散射检测器分析神经酰胺的方法并进行了含量的测定.色谱柱:ZORBZX Eclipse XDB-C18(4.6mm×250mm,5μm),洗脱方法:梯度洗脱,柱温:35℃,流动相:甲醇/水,流速:1ml/min;检测器:蒸发光散射检测器,漂移管温度:40℃,氮气流速:1.5L/min.系统探讨了梯度洗脱的起始浓度、洗脱的时间和洗脱梯度的程序设置,最佳的梯度洗脱条件为5min内,甲醇浓度从60%线性增加为90%,从5min到25min,甲醇浓度线性增加为95%,在此条件下样品和标准品的分离色谱峰对称性较好.随后测定了各种样品中神经酰胺的含量,并进行了方法学验证,结果神经酰胺在0.2～2μg之间线性关系良好,最低检测限为0.01mg/ml,R2=0.9992;平均回收率为93.3%,RSD=1.65%(n=5).本法灵敏、方便、准确,重现性好,可用于魔芋神经酰胺类物质的分离及其含量的测定.     

气相色谱法测定生物制品中苯酚含量

本文采用气相色谱法测定了几种生物制品中苯酚含量。样品无需进一步处理,加入内标液后直接上柱测定,色谱峰形良好,线性范围宽,平均回收率和变异系数分别为１０１．８％ 和１．０８５％ ,最小检测限为１０ μｇ／ｍ ｌ,本法操作简单,样品用量少,可作为测定生物制品中苯酚含量的一种常规方法。     

淡色库蚊对四种化学物质的嗅觉反应

在室内条件下通过Y型嗅觉仪测试了淡色库蚊Culexpipiens pallens雌成虫对乳酸、丙酮、氨水、辛醇、正庚酸、对甲酚、间甲酚等7种化学物质的嗅觉反应。结果表明:与对照相比,1和10mg/L氨水,1,10和100mg/L正庚酸,1mg/L辛醇,0.1和1mg/L对甲酚对淡色库蚊雌成虫具有显著的引诱作用。比较各处理的相对引诱率,最高的为10mg/L正庚酸,达50%以上;其他处理相对引诱率均低于40%,最低的为0.1mg/L对甲酚。     

4型肺炎球菌多糖丙酮酸含量核磁共振测定方法的建立及验证

目的 建立检测4型肺炎球菌多糖侧链上的丙酮酸(pyruvic acid, PA)含量的定量核磁共振(quantitativenuclear magnetic resonance, qNMR)方法，并进行验证。方法 用以3-(三甲基硅)丙酸-D4钠盐为内标的重水作为溶剂，在600 MHz核磁共振仪上采集波谱，采集参数为：脉冲程序zg30,弛豫延迟时间9 s,扫描次数512次。对方法进行线性、范围、专属性、准确度、精密度、耐用性验证。结果 PA含量在0.10～12.00 mg/mL范围内线性良好，且R²> 0.99。以PA上的甲基质子峰(化学位移1.54 ppm)为特征峰测定PA信号峰，qNMR检测方法专属性良好。两种方法的回收率分别在93.52%～101.37%和92.71%～100.10%之间；建立的方法重复性及中间精密度良好，不同PA含量测定值RSD分别为3.67%,2.12%和3.70%;改变温度和扫描次数，2种条件的测定结果差异均无统计学意义(P>0.05)。结论 建立的4型肺炎球菌多糖中PA含量qNMR检测方法简便、切实有效，为13价肺炎球菌结...     

高效液相色谱法测定17AA氨基酸注射液中N-乙酰-L-半胱氨酸和N-乙酰-L-酪氨酸

建立了一种用高效液相色谱测定 17AA氨基酸注射液中N 乙酰 L 半胱氨酸和N 乙酰 L 酪氨酸含量的方法。样品经稀释后 ,直接上机测定 ,其色谱条件为 :WatersSymmetryC18色谱柱 ,流动相为 8.5mmol/LNaAc - 5 %CH3 OH (pH =4.0 ) ,柱温36℃ ,流速 1.0ml/min ,检测波长 195nm ,N 乙酰 L 半胱氨酸在 5 .4mg/L到 5 40mg/L、N 乙酰 L 酪氨酸在 4.9mg/L到 490mg/L之间 ,标准曲线呈良好的线性关系 (r2 >0 .9999)。该方法具有良好的重现性 ,日内相对标准偏差小于 3% ,日间相对平均误差小于 7% ,样品回收率在 90 %～ 110 %之间。该方法与测定氨基酸注射液的其它高效液相色谱法相结合 ,能够对 17AA氨基酸注射液中所有氨基酸成分进行全面检测。     

化妆品中的甲硝唑、氧氟沙星与环丙沙星HPLC检测法

目的:建立反相高效液相色谱同时测定化妆品中的甲硝唑、氧氟沙星与环丙沙星方法.方法:以十八炕基硅烷键合硅胶为填充剂,以0.01mol/L四丁基溴化铵溶液(三乙胺调pH至3.0)-甲醇(88:12)为流动相,柱温30℃,流速1.0ml/min.315nm、293nm、277 nm分别作为甲硝唑、氧氟沙星、环丙沙星含量测定的检测波长.样品先用乙腈-水(0.1%甲酸)(体积比5:95)溶液提取,经正己烷脱脂后测定.结果:甲硝唑在2.02～50.59μg/ml,氧氟沙星在2.03～50.69μ g/ml,环丙沙星在2.02～50.45μg/ml范围内与峰面积线性关系良好,r分别为0.9999,0.9998和0.9999.10,25,40μ/g/ml三个浓度水平甲硝唑、氧氟沙星和环丙沙星的平均回收率为85.88%～97.20%,RSD为1.11%～6.10%;检测限均为1ng.结论:本法快速、准确、可用于化妆品中擅自添加的甲硝唑、氧氟沙星与环丙沙星的筛查及含量测定.     

蛭龙活血通瘀胶囊的质量标准研究

目的:建立蛭龙活血通瘀胶囊的质量标准.方法:采用薄层色谱法对蛭龙活血通瘀胶囊中黄芪进行定性鉴别;采用HPLC-ELSD法对君药黄芪中的黄芪甲苷进行含量测定.结果:蛭龙活血通瘀胶囊中的黄芪薄层色谱中,在与对照品相应的位置上,显相同颜色的斑点;黄芪甲苷在0.0236mg/ml～0.236mg/ml间呈良好的线性关系,r2=0.9998,样品平均回收率为99.02%(n=6),RSD为1.02%.结论:所建立的鉴别法专属性强,定量方法操作简单,精密度、重现性好,可以作为控制活血蛭龙通瘀胶囊的有效方法.     

Determination of benflumetol in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A reversed-phase high-performance liquid chromatographic method for the determination of benflumetol in human plasma is described. Benflumetol in plasma samples was extracted with a glacial acetic acid-ethyl acetate (1:100, v/v) mixture at pH 4.0. Chromatography was performed on a Spherisorb C₁₈ column using a methanol-water-glacial acetic acid-diethyl amine (93:6:1:0.03, v/v) mixture as the mobile phase and UV-VIS detection at 335 nm. The identity and purity of the benflumetol peak were carefully examined, and the internal standard method was applied for its quantitation. The absolute recovery of benflumetol in spiked plasma samples was 92.91% over the concentration range 5–4000 ng/ml. The recovery of internal standard “8212” at a concentration of 300 ng/ml in spiked plasma was 84.85%. The detection limit of benflumetol was 11.8 ng/ml. Plasma concentration-time profiles in healthy volunteer adults were measured after a single-dose oral administration of 500 mg of benflumetol. The assay procedures were within the quality control limits.     

High-performance liquid chromatography method for quantifying sphingomyelin in rat brain

A rapid, reproducible and accurate high-performance liquid chromatographic (HPLC) method for the quantitative determination of sphingomyelin in rat brain was developed and validated using normal-phase silica gel column, acetonitrile-methanol-water (65:18:17 (v/v)) at a flow rate of 1 ml/min, isocratic elution, UV detection at 207 nm and 1,2-dimyristoyl-sn-glycero-3-phosphocholine as an internal standard. Total run time was 10.0 min. The calibration curve was linear over the range of 0.025-0.4 mg/ml sphingomyelin (R2>0.99). The intra-day coefficient of variation ranged from 1.4% to 2.2%. The average inter-day coefficient of variation over a period of 4 days was 3.1%. The practical limit of detection was 0.005 mg/ml with a quantification limit of 0.01 mg/ml.     

Determination of amdinocillin in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of amdinocillin (formerly mecillinam) in human plasma and urine. The assay is performed by direct injection of a plasma protein-free supernatant or a dilution of urine. A 10-μm μBondapak phenyl column with an eluting solvent of water—methanol—1 M phosphate buffer, pH 7 (70:30:0.5) was used, with UV detection of the effluent at 220 nm. Azidocillin potassium salt [potassium-6-(d-(-)-α-azidophenyacetamido)-penicillanate] was used as the internal standard and quantitation was based on peak height ratio of amdinocillin to that of the internal standard. The assay has a recovery of 74.4 ± 6.3% (S.D.) in the concentration ranges of 0.1–20 μg per 0.2 ml of plasma with a limit of detection equivalent to 0.5 μg/ml plasma. The urine assay was validated over a concentration range of 0.025–5 mg/ml of urine, and has a limit of detection of 0.025 mg/ml (25 μg/ml) using a 0.1-ml urine specimen per assay.The assay was applied to the determination of plasma and urine concentrations of amdinocillin following intravenous administration of a 10 mg/kg dose of amdinocillin to two human subjects. The HPLC and microbiological assays were shown to correlate well for these samples.     

Assay of zofenopril and its active metabolite zofenoprilat by liquid chromatography coupled with tandem mass spectrometry

Zofenopril is a pro-drug designed to undergo metabolic hydrolysis yielding the active free sulfhydryl compound zofenoprilat, which is an angiotensin converting enzyme (ACE) inhibitor, endowed also with a marked cardioprotective activity. A simple, highly sensitive specific LC–MS–MS method was developed for the determination of zofenopril and zofenoprilat in human plasma. In order to prevent oxidative degradation of zofenoprilat and its internal standard, their free sulfhydryl groups were protected by treatment with N-ethylmaleimide (NEM), which produced the succinimide derivatives. The compounds and their corresponding fluorine derivatives, used as internal standards, were extracted from plasma with toluene. The reconstituted dried extracts were chromatographed and then monitored by a triple-stage-quadrupole instrument operating in the negative ion spray ionization mode. The method was validated over the concentration range of 1–300 ng/ml for zofenopril and 2–600 ng/ml for zofenoprilat. Inter- and intra-assay precision and accuracy of both zofenopril and zofenoprilat were better than 10%. The limit of quantitation was 1 ng/ml with zofenopril and 2 ng/ml with zofenoprilat. Extraction recovery proved to be on average 84.8% with zofenopril and 70.1% with zofenoprilat. Similar recoveries were shown by the above two internal standards. The method was applied to measure plasma concentrations of zofenopril and zofenoprilat in 18 healthy volunteers treated orally with zofenopril calcium salt at the dose of 60 mg.     

Efficient high performance liquid chromatograph/ultraviolet method for determination of diclofenac and 4'-hydroxydiclofenac in rat serum

A rapid and sensitive high-performance liquid chromatographic method was developed for determination of diclofenac and its major metabolite, 4'-hydroxydiclofenac, in serum from rats treated with diclofenac. The method is simple with a one-step extraction procedure, isocratic HPLC separation, and UV detection at 280 nm. Use of N-phenylanthranilic acid as the internal standard provided good accuracy without interference by endogenous compounds or 5-hydroxydiclofenac, another metabolite of interest. Limits of detection for diclofenac and 4'-hydroxydiclofenac were 0.0225 and 0.0112 microg/ml, respectively. Average extraction efficiencies of diclofenac, 4'-hydroxydiclofenac, and the internal standard were >/=76%. The method was applied to serum collected at 3h after rats were treated with an experimentally useful dosage range of 3, 10 and 50mg/kg diclofenac. Recovery (as a percentage of dose) for the 4'-hydroxy metabolite in serum was found to consistently average from 0.10 to 0.12% following each dosage, whereas recovery of diclofenac in serum declined from 0.45 to 0.37%. Thus, the method is suitable for measurement of a major diclofenac metabolite in experimental studies.     

High performance liquid chromatographic method for the determination of sumatriptan with fluorescence detection in human plasma

A rapid and sensitive high performance liquid chromatography (HPLC) method with fluorescence detection has been developed for the determination of sumatriptan in human plasma. The procedure involved a liquid-liquid extraction of sumatriptan and terazosin (internal standard) from human plasma with ethyl acetate. Chromatography was performed by isocratic reverse phase separation on a C18 column. Fluorescence detection was achieved with an excitation wavelength of 225 nm and an emission wavelength of 350 nm. The standard curve was linear over a working range of 1-100 ng/ml and gave an average correlation coefficient of 0.9997 during validation. The limit of quantitation (LOQ) of this method was 1 ng/ml. The absolute recovery was 92.6% for sumatriptan and 95.6% for the internal standard. The inter-day and intra-day precision and accuracy were between 0.8-3.3 and 1.1-6.3%, respectively. This method is simple, sensitive and suitable for pharmacokinetics or bioequivalence studies.     

High-performance liquid chromatographic determination of hydrocortisone and methylprednisolone and their hemisuccinate esters in human serum

A high-performance liquid chromatographic method is described for the simultaneous determination of methylprednisolone (MP) and methylprednisolone hemisuccinate (MPHS), or hydrocortisone (HC) and hydrocortisone hemisuccinate (HCHS) in human serum. Reversed-phase liquid chromatography was performed on a microparticulate C₁₈ column (Spherisorb, 5 μm) using a mobile phase of 2% glacial acetic acid, 30–35% acetonitrile, 70–65% water with ultraviolet detection (254 nm). The method uses 17α-hydroxyprogesterone as the internal standard for the determination of methylprednisolone and its hemisuccinate ester, or 11-deoxy-17-hydroxycorticosterone as the internal standard for the determination of hydrocortisone and its hemisuccinate ester. The sensitivity is 0.03 μg/ml for HC, 0.07 μg/ml for MP, 0.04 μg/ml for MPHS, and 0.10 μg/ml for HCHS, with a detection limit of 0.02 μg/ml for all four steroids. Calibration curves are linear up to 3 μg/ml for MP or MPHS (as equivalent MP) and up to 4 μg/ml for HC and 7 μg/ml (as equivalent HC) for HCHS. The pooled relative standard deviation for replicate samples for each steroid is < 7%. Plasma concentration—time curves are reported for MP and MPHS or HC and HCHS of two human subjects following intramuscular administration of 125 mg of methylprednisolone sodium succinate for injection, U.S.P., or 250 mg of hydrocortisone sodium succinate for injection, U.S.P.     

Improved method for the determination of trimethadione and its demethylated metabolite, dimethadione, in human serum by gas chromatography

An improved gas chromatographic method, involving the use of a wide-bore capillary column, for the determination of trimethadione and its only demethylated metabolite, dimethadione, in human serum is described. The results indicate that both substances and the internal standard (maleinimide) were well separated with no tailing peak. The detection limit was 10 ng/ml for trimethadione and 50 ng/ml for dimethadione. This improved method is reliable in terms of sensitivity, selectivity and reproducibility for the simultaneous determination of both compounds in human serum.     

Sensitive liquid chromatography-tandem mass spectrometry method for the simultaneous determination of paracetamol and guaifenesin in human plasma

A rapid and sensitive method for the simultaneous determination of paracetamol and guaifenesin in human plasma was developed and validated, using high-performance liquid chromatographic separation with tandem mass spectrometric detection. After extracted from plasma samples by diethyl ether-dichloromethane (3:2, v/v), the analytes and internal standard osalmide were chromatographed on a C18 column. Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI). The method was linear in the concentration range of 0.05-20.0 microg/ml for paracetamol and 5.0-2000.0 ng/ml for guaifenesin. The intra- and inter-day precision was within 14% for both paracetamol and guaifenesin. The assay accuracy was within +/-2.4% for the analytes. This is the first assay method described for the simultaneous determination of paracetamol and guaifenesin in plasma using one chromatographic run. The method was successfully employed in a pharmacokinetic study after an oral administration of a multicomponent formulation, containing 650 mg paracetamol, 200 mg guaifenesin, 60 mg pseudoephedrine and 20 mg dextrorphan.