柱前衍生反相高效液相色谱-荧光检测法测定大鼠血浆中的奈替米星

建立了一种简便、灵敏的氯甲酸芴甲酯(FMOC-Cl)柱前衍生反相高效液相色谱-荧光检测血浆中奈替米星的新方法,同时研究了其药代动力学。对色谱条件进行了优化,采用ZORBAX Eclipse XDB-C8柱(150 mm×4.6 mm,5 μm),流动相为乙腈-水(体积比为85:15),流速为1.0 mL/min,荧光检测激发波长为265 nm,发射波长为315 nm,得到奈替米星的平均加标回收率为96.62%～100.84%(n=3),对奈替米星检测的线性范围为0.045～8.88 mg/L,相关系数为0.9993,方法的日内与日间精密度分别低于3%与3.5%,最低检出限(S/N=3)与定量限(以3倍检出限计)分别为0.01和0.03 mg/L。方法简便、快速、灵敏,样品用量少(30 μL奈替米星血浆溶液已能满足该药含量的测定以及药物代谢的研究),为大鼠体内奈替米星的药代动力学研究提供了可靠的分析手段。     

双水杨醛邻苯二胺柱前衍生-高效液相色谱法测定茶叶中的Pb~(2+)

建立了利用双水杨醛邻苯二胺希夫碱(SALOPHEN)作为衍生剂结合高效液相色谱(HPLC)测定茶叶中Pb~(2+)含量的方法。Hypersil ODS2 C18反相色谱柱(250 mm×4.6 mm,5μm)为固定相,甲醇-水(80∶20,v/v)为流动相,检测波长为226 nm,反应体系pH为10.0。结果表明,样品中Pb~(2+)的线性范围为0.1~30 mg/L,线性相关系数为0.998 8,检出限为0.01 mg/L,加标回收率为91.87%~96.96%。该方法具有较高的灵敏度和稳定性,适用于茶叶中Pb~(2+)的检测。     

高效液相色谱法测定大鼠给药盐酸氟西汀后脑渗析液中的5-羟色胺

建立了高效液相色谱柱前衍生化法测定大鼠腹腔注射(i.p.)给药盐酸氟西汀前后脑渗析液中5-羟色胺(5-HT)的浓度变化情况。在大鼠给药盐酸氟西汀前和给药后不同时间点取其脑渗析液,加入衍生化试剂反应后,以乙腈-20 mmol/L醋酸盐缓冲液(pH 5.0)(体积比为45∶55)（含20 mmol/L辛烷磺酸钠）作为流动相,在Hypersil C18色谱柱 (250 mm×2.0 mm,5 μm)上进行分离,荧光检测波长为λex=330 nm,λem=455 nm。5-HT在浓度为0.25～5.0 nmol/L 范围内线性关系良好(r=0.9991);5-HT的最低定量限为0.25 nmol/L。本法准确可靠、简便,适用于生物样品中5-HT的浓度测定。     

Quantitative analysis of ripasudil hydrochloride hydrate and its impurities by reversed‐phase high‐performance liquid chromatography after precolumn derivatization: Identification of four impurities

We report the development and validation of a stability‐indicating reversed‐phase high‐performance liquid chromatography method with precolumn derivatization for the separation and identification of the impurities of ripasudil hydrochloride hydrate, a novel protein kinase inhibitor. 2,3,4,6‐Tetra‐O‐acetyl‐β‐d ‐glucopyranosyl isothiocyanate was chosen as the derivatizing reagent and triethylamine was added as catalyst. 200 μL sample solution (1 mg/mL), 600 μL derivatizing reagent (1 mg/mL), and 200 μL triethylamine solution (1%, v/v) were mixed and reacted at 40°C for 30 min. The separation was achieved on an Inertsil C₁₈ ODS‐3 (250 mm × 4.6 mm, 5 μm) column using mobile phases including 10 mmol monopotassium phosphate buffer (pH 3.0) and methanol in gradient mode. The column temperature was adjusted at 25°C and the flow rate at 1 mL/min. The detection was carried out at 220 nm. Different precolumn derivatization conditions as well as the high‐performance liquid chromatography conditions were optimized. Ripasudil hydrochloride hydrate and its four impurities were detected and quantitated, among which two new compounds were characterized. The proposed method was validated and proven to be selective, accurate, and precise and suitable for the quantitative analysis of ripasudil hydrochloride hydrate.     

微流控芯片毛细管电泳激光诱导荧光检测法测定片剂中盐酸美西律的含量

建立了微流控芯片毛细管电泳激光诱导荧光检测法测定片剂中盐酸美西律含量的方法,对衍生条件和电泳条件进行了系统的考察。盐酸美西律经异硫氰酸荧光素(FITC)40℃衍生6h,以20 mmol/L硼砂为电泳缓冲溶液,进样30s后,分离电压2000V,可在1 min内完成一次检测。方法的检出限为0.022 mg/L、线性范围0.108～1.079 mg/L、相关系数0.994,加标回收率为99.7%～102.3%,方法适用于盐酸美西律的检测和质量控制。     

超高效液相色谱-串联质谱快速测定食品中的新品种甜味剂爱德万甜

建立了超高效液相色谱-串联三重四极杆质谱快速筛查和确证食品中的新品种甜味剂爱德万甜的方法。样品经甲醇-水（50：50,v/v）超声提取,离心,取上清液过滤膜。采用Agilent SB-C18（150 mm×2.1 mm,5 μm）色谱柱分离,梯度洗脱,在电喷雾离子源的正离子电离模式下,用多反应监测（MRM）方式采集数据,根据保留时间和定性离子对进行定性筛查,根据峰面积和定量离子进行定量分析。结果表明：方法检出限（LOD）为0.03 mg/kg（信噪比S/N ≥ 3）,定量限（LOQ）为0.10 mg/kg（S/N ≥ 10）,回收率为80.3%~98.0%,在0.01~1.0 mg/L范围内均有良好的线性,相关系数r²均大于0.997。该方法快速、简便易行、灵敏、准确,适用于饮料、酸奶和果冻中的新品种甜味剂爱德万甜的批量检测。     

反相高效液相色谱法测定人肌腱中的胶原蛋白

建立了测定人肌腱中胶原蛋白含量的高效液相色谱法。动物肌腱中的胶原蛋白经酸水解后生成包括羟脯氨酸在内的氨基酸混合物,用2,4-二硝基氯苯对水解产物衍生化,然后以0.01 mol/L乙酸钠-乙酸缓冲液(pH 6.0)-乙腈(体积比为80∶20)为流动相,经反相C18柱分离,紫外检测器于360 nm波长处检测来测定羟脯氨酸的含量。羟脯氨酸是胶原蛋白的特异性氨基酸且含量稳定,因而可通过样品中羟脯氨酸的含量来计算胶原蛋白的含量。该方法对羟脯氨酸的检出限为3 μg/L,羟脯氨酸为3 μg/L~100 mg/L时与峰面积的线性关系良好;样品测定的相对标准偏差为1.95%,加标回收率为98.4%~110.8%。对60份人肌腱样品中胶原蛋白的含量进行了测定。所建立的方法灵敏、准确、干扰少,适用于肌腱中胶原蛋白含量的测定。     

A sensitive and selective determination method of histamine by HPLC with intramolecular excimer-forming derivatization and fluorescence detection

A highly sensitive, selective and simple method is described for the determination of histamine by high-performance liquid chromatography (HPLC) with fluorescence detection. The method is based on an intramolecular excimer-forming fluorescence derivatization of histamine with 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE), followed by reversed-phase HPLC. Histamine, having two amino moieties in a molecule, was converted to the dipyrene-labeled derivative by reaction with PSE. The derivative afforded intramolecular excimer fluorescence (450-540 nm), which can clearly be discriminated from the monomer fluorescence (370-420 nm) emitted from PSE. Typically, a 10 micro L sample solution was mixed with 100 micro L of derivatization reagent solution, which was a mixture of 0.5 mm PSE in acetonitrile and 0.5 mm potassium carbonate in water (8:2, v/v). The derivatization was carried out at 100 degrees C for 90 min. The PSE derivative of histamine could be separated by reversed-phase ODS column with isocratic elution using acetonitrile:water (82:18, v/v) containing 0.03% triethylamine. The detection limit (singnal-to-noise ratio = 3) of histamine was 0.5 fmol for a 30 micro L injection. The method was successfully applied to the determination of histamine in human urine, and had enough selectivity and sensitivity for urinary histamine quantification.     

反相高效液相色谱法测定人参皂甙Compound-K的含量

人参皂甙compound-K(C-K)在人参中的含量极低,但它是其他含量较高的人参皂甙Rb1和Rb2等在人体肠道内的主要 降解产物和最终吸收形式,具有很高的生物活性。采用反相高效液相色谱法测定了人参总皂甙发酵液中C-K的含量。色谱 条件为:反相C18柱;乙腈-水(体积比为48∶52)溶液为流动相,流速1.0 mL/min;紫外检测波长203 nm;柱温35 ℃;外标法 定量。结果表明:C-K的质量浓度为0.05~0.8 g/L时,其峰面积与质量浓度具有良好的线性关系,相关系数为0.9998。方法 的检测限(S/N=3)为2.5 mg/L,峰面积测定值的相对标准偏差(n=6)为2.20%。测定栽培人参总皂甙及三七茎叶总皂甙微生 物发酵液中C-K的平均加标回收率(n=3)分别为98.6%和99.7%。该方法快速简便,准确可靠,可用于C-K的制备研究及药物 开发。     

高效液相色谱法测定药品中的乙二醇二乙醚二胺四乙酸

建立了高效液相色谱分析方法用来测定药品中乙二醇二乙醚二胺四乙酸(EGTA)含量,通过检测EGTA与Cu²⁺的配合物EGTA-Cu来检测EGTA。采用Ultimate-AQ C18色谱柱(250 mm×4.6 mm, 5 μm),流动相为乙腈-四丁基氢氧化铵水溶液(质量分数约0.3%四丁基氢氧化铵水溶液,醋酸调pH 6.50)-醋酸钠溶液(35 mmol/L醋酸钠,醋酸调pH 6.50)(20:20:60, v/v/v),检测波长为245 nm,流速为1.50 mL/min,柱温为40 ℃,进样量为100 μL。结果表明,EGTA质量浓度在0.10~15.00 mg/L范围内线性关系良好(R=0.9998);以信噪比(S/N)为3及10确定检出限和定量限,分别为0.05 mg/L和0.17 mg/L;样品加标平均回收率为98.34%~99.03%, RSD为1.08%~3.33%(n=9)。该方法操作简便,具有分离度好、灵敏度高、重复性好、回收率高等特点,适合药品中EGTA含量的检测,为EGTA检测提供了一种有效的检测方法。     

离子色谱-脉冲安培电化学检测引流组织液中的妥布霉素

建立了一种离子色谱(IC)分离,脉冲安培电化学法(PAD)直接检测人体引流组织液中妥布霉素的分析方法.采用高交换容量阴离子交换柱和低浓度KOH在线淋洗分离,不需要采用衍生化和离子对-柱后加碱等实现分离,并可在短时间内完成妥布霉素的测定.采用本方法测定的妥布霉素线性范围为0.05～10 mg/L,线性相关系数为0.9997,保留时间、峰面积和峰高的相对标准偏差分别为0.14％,0.38％和0.81％,检出限为7.11 μg/L.本方法成功应用于骨髓炎患者引流组织液中妥布霉素的测定,样品实际加标回收率为100.8％.     

邻苯二甲醛柱前在线衍生反相高效液相色谱法测定功能饮料中赖氨酸的含量

以邻苯二甲醛作为衍生试剂,结合柱前在线衍生–反相高效液相色谱技术,建立了一种快速检测功能饮料中赖氨酸含量的分析方法。对流动相梯度洗脱程序、DAD检测波长和在线衍生时间等参数进行了优化。赖氨酸在1~200 mg/L范围内与色谱峰面积有良好的线性关系,相关系数大于0.998,赖氨酸的检出限为0.3 mg/L,测定结果的相对标准偏差小于7%(n=5),加标回收率为96.43%~115.01%。该方法可应用于功能饮料中赖氨酸的快速检测。     

Determination and validation of duloxetine hydrochloride in capsules by HPLC with pre-column derivatization and fluorescence detection

A high-performance liquid chromatographic (HPLC) method is described for the determination of duloxetine hydrochloride in capsules. The method was based on pre-column derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole using the fluorimetric detection technique. Duloxetine hydrochloride was analyzed by HPLC using an Inertsil C18 column (5 μm, 150 × 4.6 mm) and mobile phase consisted of methanol and water (65:35, v/v). The fluorescence detector was adjusted at excitation and emission wavelengths of 461 and 521 nm, respectively. The linearity of the method was in the range of 10-600 ng/mL. Limits of detection and quantification were 0.51 and 1.53 ng/mL, respectively. The proposed method was successfully applied for determination of duloxetine hydrochloride in its pharmaceutical preparation. The results were in good agreement with those obtained using a reference method.     

Determination of tetraethyl ammonium by ion-pair chromatography with indirect ultraviolet detection using 4-aminophenol hydrochloride as background ultraviolet absorbing reagent

A method was developed for the determination of tetraethyl ammonium (TEA) by reversed-phase ionpair chromatography with indirect ultraviolet detection. Chromatographic separation was achieved on a reversed-phase C18 column using background ultraviolet absorbing reagent - ion-pair reagent - organic solvent as mobile phase. The effects of the background ultraviolet absorbing reagents, detection wavelength, ion-pair reagents, organic solvents and column temperature on the determination method were investigated and the retention rules discussed. Results found that TEA could be successfully analyzed by using 0.7 μmol/L 4-aminophenol hydrochloride and 0.15 μmol/L 1-heptanesulfonic acid sodium mixed with 20% (v/v) methanol asmobile phase at a UV detection wavelength of 230 nm. Under these conditions, the retention time of tetraethyl ammonium was 2.85 min. The detection limit (S/N = 3) for TEA was 0.06 mg/L. The relative standard deviations (n = 5) for peak area and retention time were 0.35% and 0.02%, respectively. The method has been successfully applied to the determination of synthesized tetraethyl ammonium bromide. Recovery of tetraethyl ammonium after spiking was 99.1%.     

柱前衍生/高效液相色谱法测定果蔬中仲丁胺残留

建立了高效液相色谱(HPLC)测定仲丁胺的分析方法。试样采用全自动凯氏定氮仪蒸馏,蒸馏液经碱中和,以9-氯甲酸芴甲酯(FMOC)为衍生剂在碱性条件下衍生,衍生产物经C18柱分离,紫外检测器(265nm)检测。实验考察了衍生剂浓度、硼酸盐缓冲溶液的p H值、反应温度与时间等因素对衍生反应的影响,结果表明,最优的衍生剂浓度为0.50 g/L,缓冲溶液p H值为8.0,反应温度为室温,反应时间为15 min。在此条件下,仲丁胺在0.001~1.000 mg/L浓度范围内与其响应信号呈良好的线性关系,相关系数为0.999 8,加标回收率为82.4%~95.2%,相对标准偏差为1.3%~6.8%,方法检出限为0.1μg/kg,定量下限为0.5μg/kg。该方法快速、简便、安全、灵敏度高、重现性好,可用于果蔬中仲丁胺残留的测定。     

二维阀切换离子色谱法测定海带中游离氨基酸

建立一种测定海带中游离氨基酸的阀切换高效阴离子交换色谱耦合脉冲安培检测器法。采用一根阳离子交换柱对氨基酸进行富集,而后经阀切换至氨基酸分析柱Amino Pac~PA-10(250 mm×2 mm)上分离并进入安培检测器检测。在最佳分离条件下,20种氨基酸的质量浓度在0.1~20.0 mg/L范围内与其色谱峰面积线性关系良好,线性相关系数r~20.99,20种氨基酸的检出限为0.01 mg/L,加标回收率为83.12%~117.34%,测定结果的相对标准偏为1.02%~13.05%(n=8)。该方法样品前处理简单,无基底杂质干扰,适用于海带样品中游离氨基酸的测定。     

柱前衍生高效液相色谱法测定二乙醇胺脱氢产物亚氨基二乙酸和甘氨酸

以对甲苯磺酰氯(PTSC)为衍生剂,建立了柱前衍生高效液相色谱(HPLC)测定二乙醇胺脱氢产物中亚氨基二乙酸(IDA)和甘氨酸(Gly)含量的分析方法。IDA和Gly与衍生剂在碱性(pH 11)条件下于45℃反应15 min,进行柱前衍生,并利用高效液相色谱-质谱对衍生产物进行定性分析。衍生化产物采用VP-ODS色谱柱(200 mm×4.6 mm,5μm)分离,以0.03 mol/L醋酸铵溶液(pH 5.5)为流动相A、乙腈为流动相B(体积比为87∶13),进行等度洗脱,流速为1 mL/min,并采用配有紫外检测器的高效液相色谱仪测定,检测波长为235 nm。该法在IDA质量浓度为900~2 100 mg/L、Gly质量浓度为20~100 mg/L的范围内线性关系良好,相关系数(R2)均大于0.999。IDA和Gly的检出限(LOD)分别为0.089 7 mg/L和0.026 2 mg/L,加标回收率分别为98.7%~99.3%和98.0%~99.5%,相对标准偏差(RSD)分别为0.89%~1.23%和0.95%~1.11%(n=3)。该法具有反应条件温和、准确性高的特点,可用于工艺生产中IDA和Gly含量的测定。     

免疫亲和柱净化/柱前衍生化-高效液相色谱荧光检测法测定粮谷中的T-2毒素

建立了免疫亲和柱净化/柱前衍生化-高效液相色谱荧光检测器测定粮谷中T-2毒素含量的方法。样品经甲醇-水(体积比为80∶20)混合溶剂提取,通过免疫亲和柱(IAC)净化,以氰酸蒽(1-AN)为衍生化试剂、4-二甲基氨基吡啶(DMAP)为催化剂进行衍生,以ZORBAX Eclipse XDB-C18 柱为分离柱,乙腈-水(体积比为80∶20)为流动相进行高效液相色谱分离及荧光检测，荧光检测的激发波长为381 nm，发射波长为470 nm。T-2毒素的质量浓度为0.01~1.5 mg/L时与峰高呈良好的线性,相关系数为0.9985。在0.01~1.5 μg/g添加水平下，回收率为79.7%~94.5%,相对标准偏差小于7%；检出限（S/N＝3）为0.01 μg/g。该方法净化效果好,灵敏度高,操作简便快速。     

Derivatization of secondary amines with 2-naphthalene-sulfonyl chloride for high-performance liquid chromatographic analysis of spectinomycin

A normal-phase high-performance liquid chromatographic (HPLC) method has been developed for the assay of spectinomycin hydrochloride and spectinomycin sulfate for detection at 254 nm. The method involves pre-column derivatization of secondary amines of spectinomycin with 2-naphthalenesulfonyl chloride (NSCl) using a catalyst. Lincomycin, 1-methylpyrrole, 2-acetyl-1-methylpyrrole, and 2-acetyl-pyrrole act as catalysts for sulfonylation of spectinomycin. Without a catalyst, the derivatization reaction forms a considerable amount of actinospectinoic acid, a degradation compound of spectinomycin, and peak area:weight ratio of the derivative is approximately 15% lower than those with the catalyst. Following derivatization the sample is extracted and chromatographed on a normal-phase silica column with detection at 254 nm. The method is applicable for the analysis of both the hydrochloride and sulfate salt forms of spectinomycin. All the known degradation compounds of spectinomycin such as actinamine, actinospectinoic acid and the biosynthesis intermediates, dihydrospectinomycin diastereoisomers, are completely separated with this method. Mass spectrometric data confirms that spectinomycin is derivatized with NSCl at the secondary amines located at positions 6 and 8 of the ring structure. The standard curves for the HPLC assay of spectinomycin hydrochloride and sulfate are linear with correlation coefficients of 0.9997 and 0.9999, respectively over the range of 0.05 mg/ml to 0.3 mg/ml. The relative standard deviations (R.S.D.) of the HPLC assay methods for spectinomycin hydrochloride and sulfate are 0.67% and 0.86%, respectively. Spectinomycin hydrochloride and sulfate bulk drugs were assayed by the HPLC method and compared to gas-liquid chromatography and microbiological assay results. The HPLC method was used to assay spectinomycin in a veterinary formulation, Linco-Spectin soluble powder. The sensitivity of the HPLC assay was determined to be approximately 4 ng sample load on the column, which suggests applicability in serum and residue level studies.     

草甘膦的邻硝基苯磺酰氯柱前衍生高效液相色谱分析

以邻硝基苯磺酰氯(NBSC)为衍生化试剂,建立了柱前衍生草甘膦的反相高效液相色谱紫外检测法,并对衍生化条件进行了优化.最佳衍生化条件为:衍生温度25℃,反应时间10 min,硼砂缓冲溶液浓度0.25 mol/L(pH 9.0),草甘膦与NBSC的摩尔比为1:5.HPLC分析条件为:采用Lichrospher C18柱,...