Development and validation of analytical method for the estimation of lamivudine in rabbit plasma

Lamivudine has been widely used in the treatment of HIV disease. A reliable, sensitive reversed phase high performance liquid chromatography （RP-HPLC） method was developed and validated for lamivudine in rabbit plasma. The method was developed on Hypersil BDS C-18 column （250 mm ＊ 4.6 mm, 5 μm） using a mobile phase of 0.25% Triethylamine buffer （pH 3.0）： acetonitrile （70：30, v/v）. The efficient was monitored by UV detector at 256 nm. The total run time was 15 min with a flow rate of 1.0 mL/min. Calibration curve was linear over the concentration range of 25-2000 ng/mL. The retention times of lamivudine and internal standard （Nelfinavir） were 8.78 min and 10.86 min, respectively. The developed RP-HPLC method can be successfully applied for the quantitative pharmacokinetic parameters determination of lamivudine in rabbit model.

An Ion-Pair HPLC Method for Simultaneous Determination of Exogenous Phosphocreatine and Its Metabolite Creatine and Related ATP in Rabbit Plasma and RBC: Application to a Pharmacokinetic Study

A specific, precise and accurate ion-pair HPLC-UV method has been developed and validated for simultaneous determination of phosphocreatine (PCr), and its metabolite creatine (Cr) as well as related ATP in plasma and red blood cell (RBC) of rabbits. After addition of TMP as IS, the samples were deproteinized with 6% PCA. The analytes were separated on a Kromasil C18 column using a tertiary gradient mobile phase composed of buffer A (0.2% KH2PO4 + 0.08% tetrabutyl ammonium hydrogen sulphate, pH 3.0), buffer B (buffer A adjusted to pH 7.5 with 1 mol/L NaOH) and MeOH. Detection wavelengths were set at 210 nm for PCr and Cr and 260 nm for ATP and TMP. Some blank samples were initially run for baseline subtraction. The linear detection responses were obtained for PCr concentration over a range of 10 - 7500 mg/ml (plasma) and 5 - 2500 mg/ml (RBC) and for both Cr and ATP concentrations of 10 - 1500 mg/ml (plasma) and 5 - 750 mg/ml (RBC) (r > 0.99). The QC samples of 3 analytes showed intra-day and inter-day precisions (RSD) of - 107%. The method was successfully used to simultaneously determine plasma and RBC concentrations of the 3 analytes and to study pharmacokinetics after iv administration of PCr to rabbits.

Development and validation of a liquid chromatography with tandem mass spectrometry(LC-MS/MS) method for the determination of sunitinib in rabbit plasma

A liquid chromatography with tandem mass spectrometry（LC-MS/MS） method has been developed and validated for the measurement of sunitinib in rabbit plasma. After protein precipitation with acetonitrile, samples were analyzed on a Zorbax Extend-C18 column（150 mm×4.6 mm, 5μm）. The mobile phase consisted of a mixture of acetonitrile and deionized water（containing 0.05% formic acid） at a ratio of 27：73（v/v）, and the flow rate was set at 0.8 mL /min. The column temperature was maintained at 30 oC. The LC eluate was detected by an electrospray ionization（ESI） source operated in the positive ion mode, and quantification was conducted using MRM of the transitions m/z 399.24→283.01 and m/z 415.19→178.00 for sunitinib and internal standard（IS, diltiazem hydrochloride）, respectively. The calibration curve was linear in the range of 2–600 ng/m L. The lower limit of quantification was 2 ng/mL. The method also exhibited satisfactory results in terms of sensitivity, specificity, accuracy（with relative error ranging from –4.0% to 1.1%）, precision（with intra- and inter-day relative standard deviations ranging from 2.8% to 9.5%）, matrix effect, recovery as well as stability. Taken together, our newly developed method was reliable to monitor sunitinib concentrations in rabbit plasma.

Determination of free fatty acids in rabbit plasma by GC-MS after trimethylsilylation derivatization

In the present study, free fatty acids(FFAs, including palmitic acid, stearic acid, oleic acid, linoleic acid and arachidonic acid) in rabbit plasma were determined by gas chromatography-mass spectrometry(GC-MS) after trimethylsilylation derivatization using N,O-bis(trimethylsilyl)trifluoroacetamide(BSTFA) – trimethylchlorosilane(TMCS) as derivatization reagent. The experimental conditions, including extraction and silylation reaction, were investigated. The method was experimentally validated. The linearity between fatty acids’ peak areas and their concentrations was obtained with the corelative coefficient(r2) all more than 0.999, and the recoveries were between 82% and 111%. The intra-day variations of FFAs’ in plasma samples at different concentrations were all less than 6%. FFA analysis results of 16 rabbit plasma samples showed that the method could be well applied in the determination of plasma samples in vivo. In contrast to the traditional method of FFA derivatization, the established trimethylsilylation method presented simplicity, high specificity, and completely free from the interference of the esterified fatty acid, such as triacylglyceride. The method could be applied for analyzing FFA profiles in the clinical laboratory or pharmacological research.

A simple and sensitive gradient elution liquid chromatography with tandem mass spectrometry(LC-MS/MS) method for the quantification of doxorubicin in rabbit plasma

In the present study, we developed and validated a simple and sensitive gradient elution liquid chromatography tandem mass spectrometry （LC-MS/MS） method for quantification of doxorubicin in rabbit plasma. Daunorubicin was used as an internal standard （IS）. The doxorubicin and IS were extracted with ethyl acetate from plasma samples. The chromatographic separations were achieved on a C18 column （2.1 mm×50 mm, 2.5μm） configured with a C18 guard column （2.1 mm×10 mm, 2.5 μm）. The mobile phase of 0.1% formic acid-water solution and acetonitrile was delivered using a gradient elution program at a flow rate of 0.4 mL/min. The temperature for column was maintained at 40 ℃. The electrospray ionization （ESI） source was operated in the positive ion mode, and the quantification was conducted using multiple reaction monitoring （MRM） of the transitions m/z 544.07→396.96 and m/z 528.06→321.05 for doxorubicin and IS, respectively. The calibration curve of doxorubicin was linear （r 〉 0.999） within the range of 2-600 ng/mL. The lower limit of quantification was 2 ng/mL. The relative errors of intra-day and inter-day accuracies ranged from -2.48% to 0.18% and from -3.78% to 1.94%, respectively. The relative standard deviations of intra-day and inter-day precisions were less than 8.65% and 6.41%, respectively. The method exhibited satisfactory results in terms of specificity, sensitivity, matrix effect, recovery and stability. The newly developed LC-MS/MS method was reliable to monitor doxorubicin concentrations in rabbit plasma.

自体富血小板血浆联合自体骨移植术治疗兔桡骨骨不连实验研究

目的:观察自体富血小板血浆(platelet-rich plasma,PRP)联合自体骨移植术治疗兔桡骨骨不连的效果。方法:成年新西兰兔96只,随机分为PRP-植骨组、单纯植骨组和对照组各32只。将所有实验兔左前臂桡骨中段截除1 cm骨段,骨蜡封闭断端髓腔,制成骨不连动物模型。PRP-植骨组采用自体髂骨植入骨折端+骨折端注射自体PRP治疗,单纯植骨组仅在骨折端植入自体髂骨,对照组不作处理。术后2、4、8、12周时采用大体形态观察、X线摄片、Micro-CT检查、组织学观察及生物力学检测等手段对骨不连愈合情况进行评估。结果:大体观察、X线摄片、Micro-CT、组织学及生物力学检测结果显示,与单纯植骨组比较,PRP-植骨组术后2、4周时软骨细胞增生明显增多,术后8周时已有明显骨痂形成。PRP-植骨组术后2、4、8、12周被破坏时的最大载荷(payload-maximum,Pm)及破坏弯矩(moment,M)均高于同期单纯植骨组,差异均有统计学意义(P<0.05)。结论:自体富血小板血浆联合骨移植术治疗兔桡骨骨不连效果良好,骨愈合速度更快,骨愈合强度更高。

Desmosterol, the main sterol in rabbit semen： distribution among semen subfractions and its role in the in vitro spermatozoa acrosome reaction and motility

Sterols are essential components of the cell membrane lipid bilayer that include molecules such as cholesterol and desmosterol, which are significantly found in the spermatozoa of several animal species. However, the presence of desmosterol in rabbit semen has never been investigated. The aims of this study were to characterize the sterol composition of subfractions of ejaculated rabbit semen and evaluate the in vitro effects of sterol on the spermatozoa acrosome reaction and motility. Two sterols, occurring prevalently in the free form （94.3%）, were identified in whole semen collected from 10 fertile New Zealand White rabbits, specifically desmosterol （58.5% of total sterols） and cholesterol （35.9% of total sterols）. Desmosterol was the predominant sterol found in all subfractions of rabbit semen, varying from 56.7% （in the prostatic secretory granules, PSGs） to 63.8% （in the seminal plasma）. Spermatozoa contained an intermediate proportion of desmosterol （59.8%）, which was asymmetrically distributed between the heads （52.0% of the total content of sterols） and the tails （81.8%）. Results showed that both desmosterol and cholesterol can be transferred from the PSGs to the spermatozoa and are equally effective in inhibiting in vitro spermatozoa capacitation at a concentration higher than 1 mg L^-1. In contrast, neither desmosterol nor cholesterol had a significant effect on spermatozoa motility. Thus, it was concluded that, the various fractions of rabbit seminal fluid differ from each other in sterol composition and quantity, probably due to their different functional properties, and these fractions may undergo significant sterol changes depending on the stage of spermatozoa capacitation.

Methionine and Threonine Requirements of Dutch Rabbits Fed under a Cecotrophy Prevention Program

It is suggested that the difference in body size between domestic-type rabbits and small pet-type rabbits results in different nutrient requirements. The nutritional requirements of pet rabbits have been assessed, although such assessments require evaluation throughout the rabbit life span. The present study was conducted under a cecotrophy prevention program with young and adult rabbits. Six male Dutch rabbits were housed individually in a dormitory-type cage, and they were randomly fed graded levels of dietary methionine and threonine, at ratios of 4:0, 3:1, 2:2, 1:3, and 0:4 mixed with two types of feed, 4:0 and 0:4. Four days after switching diets and 4 hrs after starting morning feeding, approximately one milliliter of blood was collected from the vein of the ear. Free amino acid concentrations in the plasma were determined with a high-speed amino acid analyzer. Plasma concentrations of methionine and threonine compared to dietary methionine and threonine levels are shown in young rabbits. The plasma concentration of methionine remained constant at a low level and then increased linearly. The intersection was estimated as 0.16 g/d. In the same manner, the intersection of the plasma threonine value was estimated as 0.27 g/d. These values were calculated as 0.27% and 0.47% of the diets, respectively. In the case of adult rabbits, the baseline was not obtained for dietary methionine and threonine levels. The methionine requirement was estimated as 0.11 g/d. The threonine requirement was estimated as 0.22 g/d. These values were calculated as 0.15% and 0.30% of the diets, respectively. In comparison with young and adult rabbits, both methionine and threonine showed a low baseline level in young rabbits, while their variations in plasma levels of adult rabbits were not determined. The requirement of both amino acids in young rabbits is higher than that in adult rabbits. The dietary values of both amino acids in young rabbits were also higher than those in adult rabbits. Small pet-type adult rabbits required lower amino acid levels than domestic-type rabbits.

ABO血型不相容亲属活体肾移植23例报告

目的探讨ABO血型不相容(ABOi)亲属活体肾移植的临床疗效和安全性。方法回顾性分析23例ABOi亲属活体肾移植受者的临床资料。术前根据受者的初始血型抗体滴度,采取不同的个体化预处理方案,包括口服免疫抑制药+利妥昔单抗,或口服免疫抑制药+血浆置换和(或)血浆双重滤过+利妥昔单抗等,监测预处理前、后,肾移植术前及术后的血型抗体滴度和围手术期移植肾功能、相关并发症。并随访移植肾功能及相关并发症。结果23例ABOi亲属活体肾移植受者中,除1例术中出现超急性排斥反应,其余22例血清肌酐水平恢复良好。围手术期并发症包括4例淋巴瘘、1例尿瘘、1例肾周血肿合并T细胞介导的排斥反应、6例泌尿系统感染、1例急性肾小管坏死、1例急性胰腺炎、1例血型抗体反弹、1例原发病复发,经治疗均痊愈。截止至随访日,22例受者的移植物和受者存活率均为100%,移植肾功能良好。随访期间血型抗体滴度均≤1∶8。随访期并发症包括2例严重肺部感染、1例抗体介导的排斥反应、2例原发病复发、1例淋巴囊肿、1例泌尿系统感染、1例带状疱疹、1例BK病毒尿症和2例血糖异常。结论根据不同血型抗体水平选择个体化预处理方案,可以安全地实施ABOi亲体肾移植。但大剂量使用利妥昔单抗,或在高致敏受者中联合使用兔抗人胸腺细胞免疫球蛋白诱导,均可能出现严重的感染并发症。

高效液相色谱法测定家兔血浆中普萘洛尔浓度

目的建立并验证测定家兔血浆中普奈洛尔浓度的高效液相色谱方法。方法以盐酸地尔硫做内标,碱化的乙醚-正己烷提取后再用盐酸溶液反提取处理血浆样品;色谱柱为Symmetry C18柱(4.6 mm×250 mm,5μm);柱温:25℃;流动相为0.02 mol·L^(-1)磷酸盐钠盐缓冲溶液(含0.2%三乙胺,加2%磷酸溶液调节pH 4.5)-乙腈(70∶30);流速:1.0 ml·min^(-1);检测波长:290 nm;进样量:20μl。结果盐酸普萘洛尔的含量测定不受家兔血浆中内源性物质的干扰;在该方法下普萘洛尔在0.04~1.60μg·ml^(-1)的浓度范围内与峰面积线性关系良好;平均提取回收率为70.81%;日内及日间精密度RSD均小于10%。结论本研究建立的高效液相色谱方法操作简单、准确可靠、易普及,适用于家兔血浆中盐酸普萘洛尔的浓度测定。

HPLC-MS(TOF)法测定家兔血浆中黄芪甲苷的浓度

目的建立测定家兔血浆中黄芪甲苷的HPLCMS(TOF)法。方法含药血浆用C18固相萃取小柱纯化样品,用LC/MS(TOF)联用技术,以电喷雾(ESI)作为接口技术,选择黄芪甲苷的离子([M+Na]+807)和内标人参皂甘Rg1的离子([M+Na]+823)作为测定离子,测定血浆中黄芪甲苷的浓度。结果黄芪甲苷的回归方程AsAi=1.2668×103C+0.096557,r=0.999;线性范围为10～5000ng·mL-1,定量限为5ng·mL-1,回收率大于90%。结论该测定方法灵敏度高、专属性好、快速,可满足黄芪甲苷药代动力学研究要求。

内毒素诱生自由基致兔血循环系统损伤及CA对其影响

将56只健康白兔随机分组:正常对照组( 组)8只,ET处理组( 组)、CA保护组( 组)各24只,第1、2、3、4和5小时分别采集血液制备血浆样品,检测血浆T-AOC、T-SOD活性和MDA含量,第2和第5小时捕杀并迅速采集心脏制备组织匀浆,检测心脏GSH、GSH-ST、GSH-Px活性,研究内毒素(endotoxin,ET)诱导休克兔产生自由基对血浆和心脏的损伤及阳离子A(cationA,CA)对其影响。结果表明: 组各项指标均保持相对稳定; 组MDA含量比 组升高(P<0.01),T-AOC、T-SOD活性比 组降低(P<0.01),而 组的MDA、T-AOC、T-SOD显著高于 组(P<0.05);除心脏GSH-Px活性第5小时降低不明显外, 组GSH、GSH-Px和GSH-ST活性显著降低(P<0.01), 组比 组GSH、GSH-Px、GSH-ST活性高(P<0.01)。结果提示:CA能减轻ET诱生自由基所致的过氧化损伤,有效缓解内毒素引起的血液循环系统毒性损伤。

高效液相色谱法同时测定血浆中的10种蛋白同化激素

建立了一种用于10种蛋白同化激素的同时分离检测的高效液相色谱法。根据被分析物的性质，以C18反相色谱柱为分离柱，以乙腈和水为流动相，采用梯度洗脱方式，并在194—290nm的范围内快速调节检测波长，使各物质均在最大吸收波长处被检出。在优化的条件下，10种被测组分在10min内实现了快速的基线分离，检出限在0．01-0．10μg／mL范围内。在兔血浆中进行加标回收率测定．10种被测组分的加标回收率为70．3％～120％。选取美雄醇为代表进行实际动物实验，成功检测到耳脉注射美雄醇后兔血浆内的美雄醇成分。实验结果表明该方法可行，快速简便，准确可靠。

冻干兔血浆在金黄色葡萄球菌检验中的应用研究

探讨冻干兔血浆在金黄色葡萄球菌检验中的凝固性能。按照GB 4789.10-2010金黄色葡萄球菌检验方法,比较本实验室制备的冻干兔血浆与国内其它两个厂家的兔血浆对金黄色葡萄球菌标准菌株及分离菌株血浆凝固酶实验效果,并用乳胶凝集实验对冻干兔血浆检测结果进行验证。本实验室制备的冻干兔血浆的凝固性能与新鲜兔血浆相当,对金黄色葡萄球菌凝固酶阳性菌株检测特异性均为100%。本实验室制备的冻干兔血浆对金黄色葡萄球菌凝固酶的检测特异性很高,具有简便、快速等优点,其性能优于其它两个厂家。

HPLC-荧光法测定兔血浆中羟基喜树碱的浓度

目的建立高效液相色谱法（HPLC）测定兔血浆中羟基喜树碱含量的方法。方法采用HPLC-荧光法。检测器激发波长为363nm，发射波长为550nm，Liehrospher C18色谱柱，流动相为乙腈-0．075mol／L醋酸铵缓冲液（pH6．4）（32：68，含0．2％三乙胺），流速1．0mL/min，采用内标法测定浓度。结果免血浆样品中羟基喜树碱与内标（喜树碱）峰分离良好，内源性杂质不干扰样品峰，最低检测浓度为1．0μg/L。HCPT浓度范围在2．832～5800μg/L间呈线性相关，回归方程为y=0．008596＋0．004282C，相关系数为r=0．9992，相对回收率分别为100．0％-105．0％，日内和日间变异均小于15．0％。结论本实验方法具有专属性，且灵敏、可靠。适用于HCPT类制剂给药后血药浓度的检测，有助于深入研究HCPT在体内的药代动力学特征。

高效液相-荧光检测法测定兔血浆羟基喜树碱方法的研究

目的 :为了测定羟基喜树碱在人体内的血药浓度 ,建立高效液相 -荧光检测法以测定兔血浆中羟基喜树碱的含量。方法 :色谱柱为DiscoveryC18(15cm× 4 6cm ,5 μm) ,流动相为柠檬酸缓冲液 -乙腈 - 75nmol·L-1磷酸二氢钾 (70∶2 3∶7) ,75nmol·L-1磷酸二氢钾中含 1%三乙胺。流速为 1 0mL·min-1,柱温为 4 0℃ ,荧光检测波长为λex36 3nm和λem5 30nm。结果 :该方法的线性范围为 13 76 5 6～ 195 7 4 4 6 8ng·mL-1(r =0 9993) ;提取回收率和方法回收率分别为 80 90 %～ 10 3 5 9% ,10 2 72 %～ 10 8 16 % ;日内RSD≤ 7 4 9% ,日间RSD≤ 9 4 0 %。最低检测限为 5 2ng·mL-1。结论 :主效渡相—荧光法专属性强 ,重现性好 ,操作简便 ,可用于羟基喜树碱的药代动力学研究和血药浓度监测。

酮咯酸氨丁三醇在家兔体内药代动力学

目的:比较研究家兔静脉注射与局部皮肤给予酮咯酸氨丁三醇的药代动力学行为。方法:采用 HPLC 法,色谱柱为 Dia-monsil C_(18)柱(200 mm×4.6 mm,5 μm),流动相为甲醇-水-三乙胺-冰醋酸(80∶19.9∶0.02∶0.08),流速1.0 mL·min^(-1),柱温30℃,检测波长313 nm。结果:酮咯酸氨丁三醇浓度在0.1～100μg·mL^(-1)范围内与峰面积呈良好的线性关系(r=0.9981),日内 RSD 为2.1%～7.2%,日间 RSD 为8.2%～14.7%,萃取回收率为78.6%～98.6%,注射剂和凝胶剂的 T_(1/2α)分别为(0.06±0.02)h 和(1.3±0.6)h;T_(1/2β)分别为(0.6±0.2)h 和(4.4±1.7)h。结论:本文建立的方法操作简单,方法灵敏、特异,结果准确。酮咯酸氨丁三醇在家兔体内药代动力学行为符合二房室模型;用药面积差异(4倍范围内)对体内吸收量无显著影响。

家兔血浆中克林霉素的HPLC/UV检测

[目的]采用HPLC/UV法检测家兔血浆中克林霉素。[方法]血清样品经乙腈沉淀蛋白后,用二氯甲烷提取。分析柱为C18反相柱,流动相为12.5 mmol/L四丁基硫酸氢铵乙腈溶液-磷酸盐缓冲液（10 mmol/LNa2HPO4,20 mmol/LKH2PO4,pH值3.50）（20/80）,柱温40℃,流速1 ml/min。[结果]在上述色谱条件下,克林霉素血浆提取液与空白血浆中其它杂质分离良好。克林霉素血浆浓度标准曲线线性范围为40-8 000 ng/ml,血浆中定量检测限为80 ng/ml（最低）,定性鉴定限为40 ng/ml,在80-8 000 ng/ml血浆浓度范围内具有良好的线性关系。总体回收率为（93.50±0.42）%,变异系数低于4.33%。在自动样器中存放24 h、30℃保存以及反复冻融均对克林霉素的稳定性无显著影响。[结论]该方法的选择性、灵敏度和色谱分辨率均很理想,可作为检测血浆中克林霉素的一种标准方法。

诱发糖尿病饲料致新西兰兔动脉粥样硬化作用

用高脂高糖饲料喂养新西兰兔建立一种新的糖尿病并发动脉粥样硬化动物模型。将新西兰兔分为二组 :对照组 (n =7)喂普通饲料 ;实验组 (n =10 )喂含 10 %猪油、36 %白蔗糖混合饲料。共观察 2 4周 ,每 4周取禁食过夜空腹血测血糖、胰岛素、总胆固醇、高密度脂蛋白胆固醇和甘油三酯。结果发现 ,实验组动物发生高血糖和高甘油三酯血症 ,血糖浓度为 6 .36± 0 .92mmol L ,而对照组为 1.76± 0 .2 8mmol L ;实验组血甘油三酯浓度为 1.75± 0 .5 6mmol L ,而对照组为 0 .41± 0 .0 5mmol L ,两组相比 ,差异非常显著 (前者P <0 .0 1,后者P <0 .0 0 0 1)。实验结束时发现 ,实验组兔主动脉均有典型动脉粥样硬化早期病变 ,脂纹病灶面积占主动脉条展开面积的百分比为 8.5 8%±1.35 % ,而对照组仅有 0 .0 8%± 0 .0 6 % ,两组差异非常显著 (P <0 .0 0 1)。实验组动物脂纹病变主要分布于腹主动脉 ,类似于人类的动脉粥样硬化好发部位。此实验结果提示 :不含胆固醇的高脂高糖饮食可诱发高血糖、高甘油三酯血症 ,促发动脉粥样硬化 ,这是首例报告。

RP-HPLC测定家兔血浆中的儿茶素和表儿茶素

目的采用RP—HPLC法测定家兔血浆中的儿茶素和表儿茶素。方法色谱柱为Hypersi lC18柱（250mm×4．6mm，10μm），测定儿茶素和表儿茶的流动相分别为乙腈-水-三乙胺（8：92：0．3、11：89：0．3），流速均为1．0ml·min-1，检测波长分别为206、204nm，采用甲醇直接沉淀蛋白，取上清液进样。结果家兔血浆中内源性成分测定无干扰；儿茶素0．68—6．50μg·ml-1呈良好的线性关系（r=0．9994），平均方法回收率为94．7％，日内和日间RSD分别为2．72％和2．84％；表儿茶素0．25～9．92μg·ml-1呈良好的线性关系（r=0．9991），平均方法回收率为99．5％，日内和日间RSD分别为2．52％和5．92％。结论所建方法简便，灵敏度较高，结果准确，可为儿茶药材及含儿茶中药制剂的体内研究提供参考。