Determination of the unsaturated disaccharides of hyaluronic acid in equine synovial fluid by high-performance liquid chromatography and fluorescence detection

Background

The purpose of this study was to develop and validate an analytical method to determine the presence of hyaluronic acid derived disaccharides in equine synovial fluid.

Findings

A high-performance liquid chromatography method for the determination of hyaluronic acid derived unsaturated disaccharides in equine synovial fluid was developed and validated. The method is based on the measurement of unsaturated disaccharides released by digestion of linear hyaluronic acid molecules. The method showed linearity (r² = 0.996) over the full working concentration range 0.89-30 mg/l. Relative standard deviation of intra- and inter-day precision ranged from of 4.3-6.7% and 7.1-7.8% respectively. The detection limit was 0.3 mg/l corresponding to 20 mg/l in synovial fluid. Accuracy of the assay was 97-103%. This method was evaluated by determining the concentration of unsaturated disaccharides from hyaluronic acid in synovial fluid of horses with lameness in the metacarpo-/metatarsophalangeal joint localized with positive response to intra-articular anesthesia.

Conclusions

The described method is valid for determination of hyaluronic acid derived disaccharides in equine synovial fluid. This method was applied to a larger research project dealing with a new form of intra-articular therapy in horses with arthritic diseases.     

Bile acid fractionations by high-performance liquid chromatography in equine liver disease

Serum bile acids were fractionated by high-performance liquid chromatography (HPLC) in 13 control and 8 cases of liver disease in horses. The severity and type of liver injury was determined by histopathological examination of biopsy and/or necropsy specimens. The total serum bile acids (tSBA) were determined in these horses by an enzymatic method (SBA-EA) and by summation of the bile acids (SBA-LC) as fractionated by the HPLC. The SBA-LC were generally higher than the SBA-EA in both the controls and liver disease and they did not parallel each other. The primary bile acids, total cholates and total chenodeoxycholates accounted for most of the tSBA increases in liver disease. There was a shift in profile from taurocholate to free (unconjugated) cholate in direct relation to the severity of the liver injury. Among the secondary bile acids, total deoxycholates and total taurodeoxycholates increased at random. The pattern of the SBA profile in relation to the severity of the liver disease suggested that hepatocellular excretion is the most sensitive step in the enterohepatic circulation of the bile acids.     

鸡肝组织中拉沙洛西钠残留的高效液相色谱检测方法

建立了鸡肝组织中拉沙洛西钠残留的高效液相色谱检测方法.甲醇提取鸡肝组织样品中残留的拉沙洛西钠,硅胶柱净化,以磷酸盐缓冲液-乙腈-甲醇作为流动相,反相高效液相色谱-荧光检测法检测.方法平均回收率为82.1%,平均变异系数为7.75%,方法的检测限为0.02 mg/kg.     

高效液相色谱法检测饲料中咪达唑仑的含量

文中建立了高效液相色谱法测定饲料中咪达唑仑(midazolam)含量的方法。样品由乙腈提取,垂直振荡离心后使用固相萃取C18柱进行净化,色谱柱为Agilent Eclipse XDB-C18(4.6 mm×250 mm,5μm),流动相为甲醇-水梯度,检测波长254 nm。实验结果:咪达唑仑最低定量限为1 mg/kg;猪配合饲料回收率为70%～100%,猪预混合饲料回收率为60%～100%,猪浓缩饲料回收率为65%～100%;相对标准偏差均小于10%。结果提示,用高效液相色谱法测定饲料中咪达唑仑的含量,方便、简单、有效性高。     

鸡组织中尼卡巴嗪残留HPLC检测方法的研究

建立了尼卡巴嗪标识残留物在鸡组织中残留的HPLC检测方法.样品用乙腈提取,正己烷脱脂,C18固相小柱净化.以乙腈:水(52: 48)为流动相,紫外检测器在波长340 nm处检测.在该检测条件下,尼卡巴嗪标示残留物DNC的检测限为31.25μg/kg,定量限为100μg/kg.DNC工作液在31.25～8 000 μg/kg范围内药物浓度与峰面积呈良好的线性关系(r=0.999 9).尼卡巴嗪在鸡肌肉、肝脏和肾脏中添加浓度分别为0.1、0.2和0.4 μg/g时,样品回收率在80.06%～89.87%之间.批内变异系数小于7.09%,批间变异系数小于6.34%.     

Determination of bovine beta2-microglobulin and albumin in urine by a reversed-phase high-performance liquid chromatography.

Reversed-phase high-performance liquid chromatography (HPLC) was used to analyze beta2-microglobulin and albumin in bovine urine. The urine samples were chromatographed on TSK-gel ODS-120T column with an acetonitrile gradient. Urinary beta2-m and albumin were detected at 220 nm. For the pre-treatment, there were two steps proceeding injection: dialysis of urine with distilled water overnight, followed by concentration by solid-phase extraction method using a Sep-Pak cartridge. The retention times of beta2-microglobulin and albumin were 25.35 +/- 0.85 and 32.20 +/- 0.20 minutes (n=5), respectively. The mean analytical recoveries of beta2-microglobulin and albumin added to 0.1 ml of urine samples were 94.5 and 100.5%, respectively. The within-run coefficients of variation ranged from 1.5 to 5.3% for beta2-microglobulin and from 2.3 to 7.0% for albumin. The sensitivity for quantification of each protein was 0.5 microg in 100 microl injected urine samples. Urine samples from healthy cows and from cows with different types of proteinuria were analyzed by this reversed-phase HPLC. Results revealed albumin was remarkable in the urine from a cow with glomerulonephritis, and beta2-microglobulin was, in the urine from a cow with tubular dysfunction.     

Determination of uric acid in canine serum and urine by high performance liquid chromatography

A reproducible high performance liquid chromatography (HPLC) method was developed for analysis of uric acid in canine serum and urine. The method consists of precipitating serum proteins with phosphotungstic acid prior to HPLC analysis. Urine is analyzed after dilution with buffer. Chromatography is performed on a reversed-phase C-18 column with UV detection at 292 nm. Sensitivity of the method will allow reproducible measurement of uric acid at concentrations of 0.05 mg/dl in serum and 0.1 mg/dl in urine. The HPLC method has been used to quantify hundreds of canine serum and urine samples. The method is superior to UV absorption or colorimetric methods because its lower limit of detection allows measurement of uric acid at concentrations found in canine serum and urine.     

饲料中T-2毒素的高效液相色谱-荧光法检测研究

以甲醇和水(体积分数8∶2,v/v)为提取溶剂,采用硅镁吸附剂层析柱结合T-2毒素免疫亲和柱两阶段净化处理、柱前衍生,建立了饲料中T-2毒素的高效液相色谱法结合荧光检测方法。衍生液选用2-萘甲酰氯(2-NC),4-甲基氨基吡啶(DMAP)作为反应促进剂。确立的色谱检测条件为:流动相是乙腈和水(体积分数75∶25,v/v),激发波长(Ex)381 nm,发射波长(Em)470 nm,流速0.6 mL/min,柱温30℃。方法的检测限1.8 ng/g,定量限为6×103 ng/kg,标准方差(RSD)低于4.25%,回收率72.24%～83.31%,T-2毒素在25～800 ng/mL范围内线性回归良好,线性方程为:y=2186.2x-31953,标准方差R2=0.9992。本方法简单快速、灵敏、准确、重复性好,能满足饲料中检测T-2毒素的需要。     

薄层色谱法检测环丙氨嗪中5种有关物质

研究了5种环丙氨嗪有关物质和环丙氨嗪在有关物质检查薄层色谱中的响应相关性.结果表明,可以用自身对照替代对照品对照的方法对有关物质进行检测.     

高效液相色谱法测定饲料中泛酸的研究

泛酸是一种不稳定的易吸湿的油。在实际应用中以钙或钠盐形式被利用。泛酸是辅酶A（CoA）的组成部分，在动、植物组织中广泛存在，在机体的脂肪、蛋白质、糖的代谢中起重要作用。动物一旦缺乏泛酸，可使机体许多器官组织受损，出现生长缓慢，体重减轻，神经系统紊乱，出现皮肤和羽毛病变，肠道和呼吸道疾病。为此泛酸作为重要的营养素被添加到畜禽饲料中。饲料中泛酸的测定根据资料报导有紫外分光光度法、液相色谱法、气相色谱法和微生物法等。当饲料中泛酸含量偏低时，AOAC最经典的方法是采用微生物法。目前液相色谱法应用较为广泛，本…     

Identification of metoclopramide metabolites in the urine of cattle by gas chromatography-mass spectrometry and high-performance liquid chromatography-photodiode array detection

The urinary metabolites of metoclopramide (4-amino-5-chloro-N-[2-diethylaminoethyl]-2-methoxybenzamide) were identified in cows. The drug was administered intravenously, voided urine was collected, and individual urine extracts were analysed by gas chromatography-mass spectrometry and high-performance liquid chromatography-photodiode array detection. The parent compound and one major metabolite (4-amino-5-chloro-N-[2-(ethylamino)ethyl]-2-methoxybenzamide) were common to all individuals. In addition to the parent and major metabolite, a second, minor metabolite was identified in two cows as 4-amino-5-chloro-N-[2-(diethylamino)ethyl]-2-hydroxybenzamide. The identity of the minor metabolite was confirmed by comparison with a standard synthesized by a new method. Metabolite identification and characterization in food animal species allows the design of safety and environmental impact studies and relative metabolite ratios between dose treatment groups.Abbreviations ¹H-NMR proton nuclear magnetic resonance - R _T retention time - D₁ dopamine-1 - D₂ dopamine-2 - 5-HT₃ 5-hydroxytryptamine - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - UV-vis ultraviolet-visible - ID internal diameter - m/z mass/charge ratio     

Simultaneous determination of tetrahydrofolate and N5-methyltetrahydrofolate in pig plasma by high-performance liquid chromatography with electrochemical detection.

An analytical method for measurement of tetrahydrofolate (THF) and N5-methyltetrahydrofolate (5MF) in pig plasma by the high performance liquid chromatography and electrochemical detection is described. The plasma sample was deproteinized by perchloric acid and the supernatant was analyzed using the following conditions; (a) phenyl bonded phase column as an analytical column; (b) mobile phase consisting of 20 mM acetate buffer (pH 3.6) containing 0.1 mM EDTA and acetonitrile (96.5:3.5, v/v); (c) an applied potential of +300 mV. Under the above condition, both peaks of THF and 5MF in the plasma were well separated. The detection limits of THF and 5MF were 0.15 and 0.13 ng/ml, respectively, at S/N = 3. The recoveries of THF and 5MF from the plasma spiked with standard THF and 5MF were 77.6 +/- 2.1% and 83.0 +/- 1.7% (mean +/- S.D.), respectively.     

Quantitation of meloxicam in the plasma of koalas (Phascolarctos cinereus) by improved high performance liquid chromatography

An improved method to determine meloxicam (MEL) concentrations in koala plasma using reversed phase high performance liquid chromatography equipped with a photo diode array detector was developed and validated. A plasma sample clean-up step was carried out with hydrophilic-lipophilic copolymer solid phase extraction cartridges. MEL was separated from an endogenous interference using an isocratic mobile phase [acetonitrile and 50 mM potassium phosphate buffer (pH 2.15), 45:55 (v:v)] on a Nova-Pak C₁₈ 4-µm (300 × 3.9 mm) column. Retention times for MEL and piroxicam were 8.03 and 5.56 min, respectively. Peak area ratios of MEL to the internal standard (IS) were used for regression analysis of the calibration curve, which was linear from 10 to 1,000 ng/mL (r² > 0.9998). Average absolute recovery rates were 91% and 96% for MEL and the IS, respectively. This method had sufficient sensitivity (lower quantitation limit of 10 ng/mL), precision, accuracy, and selectivity for routine analysis of MEL in koala plasma using 250-µL sample volumes. Our technique clearly resolved the MEL peak from the complex koala plasma matrix and accurately measured MEL concentrations in small plasma volumes.     

Determination of selamectin in dog plasma by high performance liquid chromatography with automated solid phase extraction and fluorescence detection

A method is described for the determination of selamectin in dog plasma, using High-Performance Liquid Chromatography (HPLC) with fluorescence detection (excitation and emission wavelengths fixed at 355 and 465 nm, respectively). The fluorescent derivative was obtained by condensation reaction with trifluoroacetic anhydride and N-methylimidazole. The method employs 1 mL plasma samples and gives linear calibration graphs (r = 0.999) over the concentration range studied (0.5-50 ng mL(-1)). Automatic solid phase extraction using the benchmate procedure was used for sample preparation. This method permits the determination of selamectin at levels as low as 0.1 ng mL(-1) and its suitability was demonstrated by a pharmacokinetic study on a dog receiving the therapeutic dose (Spot-on administration).     

薄层色谱和高效液相色谱联合检测玉米赤霉烯酮的方法研究

建立了薄层色谱法(TLC)定性检测和高效液相色谱法(HPLC)定量检测相结合的检测玉米粉中玉米赤霉烯酮(ZON)的方法。以石油醚∶乙醚∶冰醋酸(70∶28∶2)为展开剂,TCL定性检测ZON,ZON的比移值(Rf值)约为0.26。HPLC对ZON进行定量分析,ZON在0.25~5μg/mL范围内线性关系良好,回收率高,检测限为3μg/kg。试验表明:TLC快速、简便,可作为大批量、快速检测ZON的有效方法;HPLC精确、灵敏度高可作为定量检测ZON的有效手段。     

Natural deoxynivalenol (DON) contamination of wheat samples grown in 1998 as determined by high-performance liquid chromatography

A high-performance liquid chromatography--diode array detection (HPLC-DAD) method was developed for determining the deoxynivalenol (DON) content of wheat and other cereals. The samples were extracted with a mixture of acetonitrile and water (84 + 16). Part of the extract was evaporated and purified on Florisil and activated charcoal columns. HPLC separation was performed on a C18 column, using acetonitrile-water (8 + 92) as eluent. Diode array detection (DAD) was performed at 218 and 236 nm, by determination of the UV spectrum. Quantitative analysis was carried out by the external standard method, using the UV spectrum obtained by DAD for confirmation. The recovery rate of DON was 75 +/- 3.1% and the detection limit was 0.05 mg/kg DON. Using this method, the DON content of 99 feeding wheat samples grown in the northeastern part of Hungary in 1998 was determined. Eighty-eight percent of the samples originating from three counties contained 0.94 mg/kg DON on the average. The highest individual value was 4.3 mg/kg. DON contamination of wheat was of higher prevalence (100%) and severity (0.27-4.3 mg/kg) in the southeastern county of Békés than in Szabolcs county located in the northeastern part of Hungary (ratio of positive samples: 82%; DON concentration: 0.05-1.3 mg/kg). The higher than usual DON contamination of feeding wheat can be explained by the rainy summer weather. DON contamination of feeding wheat poses a major risk to the production and animal health status of pig herds.     

采用全自动在线固相萃取-高效液相色谱法检测畜禽产品中氟喹诺酮类药物残留量

为建立畜产品中6种氟喹诺酮全自动在线固相萃取-高效液相色谱的检测方法,实验采用全自动在线固相萃取-高效液相色谱替代了前处理使用固相萃取小柱净化过程,净化效果较好。实现了恩诺沙星、环丙沙星、洛美沙星、培氟沙星、诺氟沙星和丹诺沙星6种氟喹诺酮类药物同时检测。本方法采用1%乙酸乙腈溶液提取,稀释后使用全自动在线固相萃取-高效液相色谱仪检测。6种氟喹诺酮平均回收率为81.9%~106.8%;相对标准偏差小于3.52%;定量限均为2.5μg/kg。该方法操作简单、经济、灵敏度高,结果准确可靠。     

高效液相色谱法检测蓖麻粕中的蓖麻碱

本试验建立了蓖麻粕中蓖麻碱的甲醇提取和高效液相色谱测定方法。蓖麻粕经甲醇80℃水浴回流提取,提取时间在2h之后蓖麻碱的得率保持稳定。使用AtlantisTMdC18色谱柱(5μm,4.6×150mm),乙腈和水(15 85)为流动相,1.0ml/L的流速等度洗脱10min,紫外检测波长308nm,经0.45μm滤膜过滤的蓖麻粕甲醇提取液中蓖麻碱得到完全分离。方法在考察的蓖麻碱质量浓度范围内为(0.005￣500μg/mL)保持良好线性,r2>0.99,实际样品测定时蓖麻碱的检出限不大于0.5μg/g。     

免疫亲和色谱柱-高效液相色谱法测定饲料中赭曲霉毒素A的研究

本文建立了饲料中赭曲霉毒素A(OTA)的免疫亲和柱净化-高效液相色谱荧光检测方法。样品用甲醇-水(体积比80∶20)提取,通过免疫亲和柱富集和净化,以乙腈和2%乙酸溶液为流动相(体积比44∶56),Agilent C18色谱柱分离,荧光检测器检测(λex-332 nm,λem=460 nm)。OTA的线性范围为0.1～100.0 ng/mL,r=0.99989。方法检测限为0.2μg/kg,定量限为0.6μg/kg。样品中赭曲霉毒素A的加标回收率为75%～92%,相对标准偏差为1.65%～3.61%。