RP-HPLC测定兔血清和脑脊液中甲硝唑含量

 报告了用RP-HPLC测定免血清和脑脊液中甲硝唑浓度的方法，采用ZorbaxC8柱，NaH2PO4水溶液（0.0075mol／L）-甲醇（4:1，PH4.0）为流动相，氢氯噻嗪为内标，检测波长320um。线性范围0.5～80mg／L，最低检出浓度80μg／L（信噪比为3）。     

糊精介质中西酞普兰的毛细管电泳手性分离与定量测定

以糊精作为毛细管电泳手性分离选择剂,对药物西酞普兰对映体的分离进行研究。考察了糊精浓度、缓冲液体系离子强度和pH及分离电压对对映体分离的影响。在糊精7．0％（m／V）、磷酸盐80mmol／L（pH5．4）的运行缓冲液中,分离电压20kV时,西酞普兰对映体分离度达3．9,同时对拆分机理进行了初步探讨。测定S-（＋）-西酞普兰原料药中R-（-）异构体的含量,在0．05～4．00g／L浓度范围内线性关系良好,R-（-）．西酞普兰与S-（＋）-西酞普兰的检出限分别为25．3mg／L和27．3mg／L,线性相关系数均在0．9970以上;RSD低于3．2％。     

固相微萃取-高效液相色谱联用测定环境水样中双酚A的自由溶解态浓度

应用可忽略耗损固相微萃取与高效液相色谱联用技术测定了环境水样中双酚A的自由溶解态浓度。为了获得高的灵敏度并减小环境因素（如温度和搅拌等）的影响,采用商品化固相微萃取纤维CW／TPR进行平衡采样。在环境水样常见pH（5～8）、缓冲容量（5～200mmol／L）和盐度（0～500mmol／L）条件下,4h可以达到萃取平衡。100mL样品足以避免样品耗损。以配制在250mmol／L NaCl和125mmol／L磷酸盐溶液（pH6．4）中的双酚A标准溶液进行校准,可以将缓冲液（0～200mmol／L）、盐度（0～500mmol／L）和pH（5．7～8．5）的影响控制在15％偏差范围以内。如需更准确的测定,也可以对样品pH值的影响加以校正。pH为6．4时,方法的线性范围为0．1～250μg／L,检出限为0．03μg／L,相对标准偏差（5μg/L,n=3）为1．1％。采用本方法测定了污水处理厂排水口的双酚A的自由溶解态浓度。     

石英芯片电泳分离检测尿中蛋白的研究

通过对缓冲体系、缓冲液浓度、酸度、乳酸钙浓度、乙胺浓度、电泳电压和进样时间的优化选择，用石英芯片电泳一紫外检测法分离了纯人白蛋白和人运铁蛋白；在75mmol／L硼酸盐（pH10．55）（含0．8mmol／L乳酸钙、1％（φ）乙胺）运行缓冲液中，上述两组分在3min内完全分离；纯人白蛋白和人运铁蛋白的线性范围分别为1．0～15．0g/L和1．0～10．0g／L；检出限（S／N=3）均为0．5g／L，应用于临床尿蛋白分离测定，并与Helena琼脂糖凝胶电泳仪电泳结果进行比较，获得一致结果。     

毛细管电泳法测定单胺氧化酶活性

应用毛细管电泳技术,建立了快速测定单胺氧化酶（MAO）活性的方法。研究对分离缓冲液pH值、浓度、毛细管表面改性剂十四烷基三甲基溴化铵（TTAB）浓度等影响因素进行优化,探讨了方法的可行性,确立了最佳分离条件。以70cm×50μm（i．d．）未涂敷熔融石英毛细管为色谱分离柱,运行电压15kv,运行缓冲液：0．5mmol／LTTAB,磷酸盐缓冲液（50mmol／L,pH 10．5）;紫外检测波长：214nm。MAO催化犬尿胺（Kyn）反应的产物4-羟基喹啉（4-HQ）的浓度和峰面积的线性范围为5～1000μmol／L,相关系数为0．9997,相对标准偏差（RSD）小于5．6％（n=5）,检出限为2μmol／L（S／N=3）。实验证明,此方法可以用于生物样品中MAO催化活性的检测。     

反相高效液相色谱法测定人血浆中利培酮及其代谢物的质量浓度

建立了测定人血浆中利培酮及其活性代谢物9-羟利培酮质量浓度的反相高效液相色谱方法。用Zor－baxODSC18色谱柱,以V（甲醇）：V（水）：V（1mol／L醋酸铵）：V（3mol／L氨水）＝300：50：3：1为流动相,检测波长为280nm,流速为0．8mL／min。利培酮的线性范围为2～600μg／L（r＝0．996）,回收率为（98．2±3．5）％,日内与日间的标准偏差分别为4．12％和4．83％;9-羟利培酮的线性范围为2～800μg／L（r＝0．998）,回收率为（97．8±3．8）％,日内与日间的标准偏差分别为4．28％和4．81％。     

离子交换色谱法检测离子液体中阴离子

建立了用阴离子交换分离柱、化学抑制模式、电导检测测定系列离子液体中BF4^-阴离子及其他杂阴离子（F^-、Cl^-、Br^-）含量的方法，并用于在线监控离子液体合成工艺中阴离子杂质含量。确定淋洗液组成为1．6mmol／L Na2CO3＋3．9mmol／L NaHCO3，流速为0．6mL／min。本方法对所测阴离子检出限分别为50μg／L（F^-、Br^-）和80μg／L（BF4^-）；线性范围在3个数量级以上；r〉0．999；回收率在98％～102％之间。方法用于对离子液体小试工艺样品分析及过程监控时，结果满意，样品的RSD小于2．6％（n=6）。     

高效液相色谱法对吗啡、杜冷丁、安定的同步测定

利用高效液相色谱法同时分析测定了血液中吗啡、杜冷丁、安定的质量浓度。分析柱为C18,流动相为V（甲醇）：V（25mmol／LKH2PO4）＝90：10,检测波长为285nm。样品血液pH8．5～9．4时,用V（氯仿）：V（异丙醇）＝9：1溶液提取,以氮气吹干有机溶剂后用流动相溶解残渣进样分析。线性范围为0．05～50mg／L,最小检出质量浓度为0．05mg／L,日内与日间精密度CV＜6％。     

流动注射化学发光法测定己烯雌酚

基于己烯雌酚对鲁米诺与高碘酸钾在碱性介质中反应产生的化学发光信号有较强的增敏作用,建立了测定己烯雌酚的流动注射化学发光分析法。己烯雌酚质量浓度在13．4—1340μg／L范围内与化学发光强度呈良好的线性关系,检出限（3d）为4μg／L;对26．8μg／L己烯雌酚进行11次平行测定,相对标准偏差（RSD）为1．4％。该法已用于药物制剂中己烯雌酚的测定。     

大豆分离蛋白甲基丙烯酸接枝共聚物的合成与表征

以过硫酸铵为引发剂、2-巯基乙醇为蛋白质变性剂,在浓度为8mol／L尿素溶液中进行了大豆分离蛋白与甲基丙烯酸的接枝共聚反应;傅里叶红外光谱和核磁共振谱证明,甲基丙烯酸成功地接枝到了大豆分离蛋白上;相关影响因素的单因数实验研究表明,各因数的最佳值分别为：引发剂浓度16mmol／L,单体浓度2．4mol／L,反应温度80℃,反应时间4h,巯基乙醇浓度0．05mol／L。     

Gas Chromatographic Determination of Ethanol in Rabbit Arterial Serum

Abstract

A glc method utilizing a flame ionization detector was developed for determining ethanol (I) in rabbit arterial serum. Addition of triton X-100 solution containing the internal standard, acetonitrile (II) to rabbit serum was followed by direct injection glc analysis using Porapak Q as the stationary phase. Ethanol was clearly separated from its major metabolite acetaldehyde (III) although the assay was not suitable for determination of III. Accuracy and precision of the method was demonstrated over the concentration range anticipated in acute intravenous dosing of I to rabbits. The method was evaluated in a rabbit which received three different intravenous infusion doses of I at two week intervals and found to be applicable to arterial serum analysis.     

凝血酶时间法测定家兔尿液及胆汁中r-水蛭素

建立了一种生物测定法---凝血酶时间法(TT法)以测定家兔尿液及胆汁中的r-水蛭素(r-Hirudin,rH),从而为rH的排泄动力学研究提供测定方法学。本方法依据rH的抗凝血酶活性,以logTT延长率对rH浓度构建标准曲线。家兔尿液或胆汁10μL,依次加入40μL空白兔血浆、50μL Tris-HCl缓冲液(pH7.4),以及50μL浓度为5U/mL的凝血酶溶液,由血凝仪记录凝血时间并计算凝血时间延长百分率,从标准曲线求出待测尿液及胆汁中的rH浓度。TT延长百分率(y)经对数转换与尿液或胆汁中rH浓度(x,mg/L)呈线性相关(r>0.99),线性范围0.49~2.50mg/L;检出限为0.49mg/L。高、中、低三种浓度rH尿及胆汁标准溶液日内及日间精密度(RSD)均<10%,方法回收率>95%,稀释回收率>91.9%,室温及-20℃保存至少8h及半月内稳定。应用上述方法测定家兔静脉注射rH后尿及胆汁药物浓度以及研究尿药排泄动力学,均表明本研究建立的TT法精密度和回收率高,结果准确可靠,符合药动学要求。     

Development and validation of a liquid chromatography/electrospray ionization tandem mass spectrometry method for the quantification of latanoprost free acid in rabbit aqueous humor and ciliary body

A rapid, selective and sensitive method for quantification of latanoprost free acid in rabbit aqueous humor (AH) and ciliary body (CB) using reverse phase-high performance liquid chromatography coupled with electrospray ionization (ESI)-mass spectrometry/mass spectrometry has been developed and validated. Quantification in AH and CB was achieved by stable isotope dilution employing tetra-deuterated analog of latanoprost free acid, used as internal standard. Sample preparation was based on protein precipitation with methanol in AH, and on liquid extraction with a mixture of ethyl acetate and isopropanol 60:40 (v/v) in CB. Elution was achieved on an octylsilica (C8) column, using an isocratic elution method. Detection was performed on a triple quadrupole mass spectrometer, using ESI in positive ion selected reaction monitoring mode. Calibration curves were linear in the validated concentration ranges of 10-160 ng/mL in AH and 80-1280 ng/g in CB. The accuracy and precision values, obtained from three different sets of quality control samples, each analyzed in triplicate on three different days, were within the generally accepted criteria for analytical methods (< 15%). The limit of detection was 30.66 pg/mL in AH and 237.75 pg/g in CB. The assay proved to be accurate and precise when applied to the in vivo study of latanoprost free acid in rabbit AH and CB after single administration of an eye drops containing latanoprost.     

凝血酶时间法测定家兔血浆中重组水蛭素

建立了凝血酶时间(thromb in tim e,TT)法测定国产重组水蛭素血药浓度的方法,以便为进一步研究该药在家兔体内的药物代谢动力学提供方法学基础。重组水蛭素血浆水平依其抗凝血酶活性(TT延长)予以测定。对该法的影响因素进行了探讨,最适测定条件确定为5 U/mL的凝血酶溶液,0.1 mol/L pH 7.4的Tris-HCL缓冲液及血浆各50μL;结果发现对凝血酶时间延长百分率的对数与浓度呈线性相关,r为0.98~0.99;线性范围为0.12~0.60 mg/L,定量限为0.12 mg/L。日内和日间精密度(RSD)分别为5.6%~9.1%和9.2%~11.5%;方法回收率97.5%~99.2%;超出线性范围的高浓度血浆样品作2~100倍稀释,其稀释回收率为92.4%~94.8%。血浆样品在-20℃冰冻保存,至少1星期内稳定。应用本方法测定家兔iv重组水蛭素2.0 mg/kg后不同时间血药浓度,表明该药符合二室模型、一级动力学过程。     

DSC measurement of cartilage destruction caused by septic arthritis

Summary Treatment of a bacterial arthritis is a challenging task for a clinician as inadequate therapy can cause cartilage destruction and can result in severe osteoarthritis of the affected joint. The development of cartilage destruction in septic arthritis is not known in details. The aim of this study was to follow this process by calorimetric method. We induced experimental septic arthritis in knee joints of seven New Zealand rabbits by single inocculation of Staphylococcus aureusOKI 112001 culture (1.5 mL 8·10⁸±5% c.f.u.). The first rabbit died on the 11^thday. At that time all the other subjects were made overslept and samples were isolated from the cartilage of the femurs for calorimetric measurement. The DSC scans clearly demonstrated the development of infective structural destruction in cartilage from the first to the tenth day of incubation. In case of healthy control the melting temperatures (T_m) were: 49.7, 55 and 63.4°C and the total calorimetric enthalpy change (ΔH) was 0.55 J g^-1. After the first day the enthalpy decreased (0.375 J g^-1), the first two transition temperature shifted towards higher temperature: 57 and 63.15°C. Up to the fourth day the effect of infection culminated with T_mof 49.3, 55.9, 59.4, 62.8°C and further decrease of the ΔH. At the fifth day the effect of infection is culminated in two separable thermal denaturation events (with 55 and 63.3°C T_ms) with high jump in ΔHindicating the dramatic change of the structure of rabbit cartilage, so this time elapsed seems to be critical from the point of view of practical clinical relevance too. Between the 7^thand 11^thdays practically we had same melting temperatures (50 and 63°C) with low (~0.24 J g^-1) enthalpy.     

高效液相色谱法测定家兔血清中盐酸山莨菪碱含量

rapid method for the separation and determination of anisodamine hydrochloride in serum of rabbit has been developed with Nucleosil column of 4. 6× 250mm and CH3OH-H2O-(C2H5)3N (30:70 : 0.125)as mobile phase. Anisodamine hydrochloride was determined by reversed phase HPLC at 214nm.Atropine sulfate was used as an internal standard. The calibration curve was Y= 0.01945X+0. 05623,r= 0. 9998,n= 6.It was rectilinear within the range between 0. 75μg/mL-50.0μg/mL,and the recovery was from 97. 0% to 98. 2%.     

Development of rapid and high‐throughput LC–MS/MS method for quantification of olprinone in rabbit plasma

A simple, highly sensitive and rapid method for quantification of olprinone (phosphodiesterase 3 inhibitor) in rabbit plasma using liquid chromatography–tandem mass spectrometry with electrospray was developed. An aliquot of 50 μL of plasma sample was cleaned up and extracted using Ostro? 96‐well plate followed by dilution. Chromatographic separation of olprinone and olprinone‐d3 was carried out on a CORTECS^® T3 column within 3 min. Detection was achieved using a triple quadrupole mass spectrometer employing electrospray ionization operated in positive ion multiple reaction monitoring mode using the transitions m/z 251.07 → m/z 155.06 and m/z 254.21 → m/z 158.10 for olprinone and olprinone‐d3, respectively. The method was validated according to US Food and Drug Administration guideline for bioanalytical methods, and showed excellent linearity in the range 10.0–2000.0 ng/mL with coefficient of determination >0.99. The intra‐ and inter‐day precisions (CV) were <5.1% and the accuracies were within the range 99.7–103.2% at all quality control concentrations. Furthermore, olprinone was stable under various stability conditions. The developed method was used for quantification of olprinone in rabbit plasma after its intravenous administration at the dose of 1 mg/kg in order to better understand the metabolism of olprinone in a rabbit model of lung injury.     

流动注射协同增强化学发光免疫分析法的建立与评价

以兔IgG为模型 ,NaTPB与PPP协同增强的Luminol H2 O2 HRP混合液为发光体系 ,HRP标记抗体 ,建立了灵敏度高、特异性强、重现性好的流动注射协同增强化学发光免疫分析法。结果表明 :在 2～ 6 0 μg L范围内兔IgG量与发光强度有良好的线性关系 ,相关系数r=0 .994 1 (P <0 .0 1 ) ;绝对检测限为 0 .6 5fmol;方法精密度为 4 .72 %～ 9.31 % ;回收率为 92 .5 0 %～ 99.4 0 %。免疫柱可反复使用 2 0 0次以上。本法测定不同浓度兔IgG标准溶液的结果与RIA法的测定结果基本一致     

Development and validation of a UPLC‐UV method for the determination of daptomycin in rabbit plasma

Daptomycin (DPT) is a lipopeptide antibiotic with potent bactericidal activity in vitro against Gram‐positive bacteria, which has attracted the attention of the scientific community due to its unique mechanism of action and due to the immediate need for new antibiotics in the era of multidrug resistance. In order to assess its pharmacokinetics in rabbits a new analytical method has been developed and validated using ultra performance liquid chromatography in conjugation with ultraviolet detection for the quantitation of the antibiotic in rabbit plasma, using the internal standard methodology. The separation was achieved employing a C₁₈ column with gradient elution using 0.1% aq. trifluoroacetic acid and methanol. The total analysis time was 2.5 min. The sample pretreatment employed protein precipitation with acetonitrile–methanol mixture and centrifugation. The method was validated in terms of linearity, precision, accuracy, sensitivity, robustness, short‐term and freeze–thaw stability and was applied to the quantification of DPT in plasma samples obtained from rabbits treated with 25 mg kg^?1 DPT. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous quantification of candesartan and irbesartan in rabbit eye tissues by liquid chromatography–tandem mass spectrometry

Diabetic retinopathy is a major cause of vision loss in adults. Novel eye-drop formulations of candesartan and irbesartan are being developed for its cure or treatment. To support a preclinical trial in rabbits, it was critical to develop and validate a new LC–MS/MS method for simultaneous quantification of candesartan and irbesartan in rabbit eye tissues (cornea, aqueous humor, vitreous body and retina/choroid). Eye tissue samples were first homogenized in H₂O-diluted rabbit plasma. The candesartan and irbesartan in the supernatants together with their respective internal standards (candesartan-d₄ and irbesartan-d₄) were extracted by solid-phase extraction. The extracted samples were injected onto a C₁₈ column for gradient separation. The MS detection was in the positive electrospray ionization mode using the multiple reaction monitoring transitions of m/z 441 → 263, 445 → 267, 429 → 207, and 433 → 211 for candesartan, candesartan-d₄, irbesartan and irbesartan-d₄, respectively. For the validated concentration ranges (2–2000 and 5–5000 ng/g for candesartan and irbesartan, respectively), the within-run and between-run accuracies (% bias) were within the range of −8.0–10.0. The percentage CV ranged from 0.6 to 7.3. There was no significant matrix interference nor matrix effect from different eye tissues and different rabbits. The validated method was successfully used in the Good Laboratory Practice (GLP) study of rabbits.