Analysis of coenzyme Q(10) in human plasma by column-switching liquid chromatography

A new method of determining coenzyme Q10 in human plasma was developed based on column-switching high performance liquid chromatography (HPLC). CoQ10 was quantitatively extracted into 1-propanol with a fast one-step extraction procedure, after centrifugation, the supernatant was cleaned on an octadecyl-bonded silica column and then transferred to reversed-phase column by a column-switching valve. Determination of CoQ10 was performed on a reversed-phase analytical column with ultraviolet detection at 275 nm and the mobile phase containing 10% (v/v) isopropanol in methanol at a flow-rate of 1.5 ml/min. The sensitivity of this method allows the detection of 0.1 microg/ml CoQ10 in plasma (S/N=3). The linearity between the concentration and peak height is from 0.05 to 20 mg/l. The reproducibility (R.S.D.%) of the method is less than 2% (within day) and less than 3% (between day), the average recovery is 100.9 + 2.1%, it takes only 30 min to complete an analysis procedure, suitable for the determination of CoQ10 in human plasma especially for batch analysis in clinical laboratories. Finally, the method was applied to determine the plasma CoQ10 levels in healthy subjects, hyperthyroid and hypothyroid patients.     

Effect of Water Stress on Nodule Physiology and Biochemistry of a Drought Tolerant Cultivar of Common Bean (Phaseolus vulgarisL.)

Plants of EMGOPA-201, a drought tolerant cultivar of commonbean(Phaseolus vulgaris), were maintained either at 90% soilfield capacity (SFC) or stressed by reducing SFC to 70, 50 or30% over a 10 d period. Plant dry weight was not affected byany of these treatments although the number and weight of noduleswas reduced at 50 and 30% SFC. Nitrogenase activity, determinedby the acetylene reduction assay (ARA), was also reduced, ona plant basis, at 50% SFC and was almost stopped at 30% SFC.The latter treatment caused a marked increase in nodule O₂diffusionresistance and induced nodule senescence. A time-course analysisof the 10 d 30% SFC treatment showed a decrease in leaf waterpotential from -0.5 to -0.87 MPa by 8 d, with a cessation ofdry weight increase after 3 d, when leaf water potential was-0.65 MPa. Proteins in the host plant fraction of nodules decreasedto 50% of control values by 10 d and leghaemoglobin (Lb) contentwas also lower at this stage. The activity of sucrose synthase(SS) showed a 76% reduction between 3 and 6 d, whilst glutamatesynthase (GOGAT) activity showed a 40% reduction. The activityof other key enzymes of carbon metabolism was also reduced after10 d. Nodule sucrose content increased to double that of controlnodules by 6 d, before declining back to control levels at 10d. Starch content fell by 3 d and continued to fall throughoutthe stress period. The results are discussed in terms of droughttolerance strategies in relation to growth and metabolism inwhole plants and nodules.Copyright 1999 Annals of Botany Company. Phaseolus vulgaris,common bean, water stress, nitrogen fixation, oxygen diffusion, acetylene reduction, enzyme activity, carbon metabolism.     

Use of a new pirkle-type chiral stationary phase in analytical and preparative subcritical fluid chromatography of pharmaceutical compounds

Good results have been obtained with use of the new bonded chiral stationary phase Whelk-O 1 in analytical and preparative subcritical fluid chromatography. A wide variety of enantiomeric pairs of compounds with different functional groups that are of pharmaceutical and biological interest have been resolved. This Pirkle-concept CSP appears to be more rugged than cellulosic phases (e.g., Chiralcel) with regards to solvents and pressure. In comparing the usefulness of the column for SFC versus HPLC chiral analysis, we have observed a clear superiority of SFC in terms of higher speed and efficiency of analysis, and faster method development. This is consistent with our experience with Chiralcel CSPs. With the Whelk-O 1 we have shown that the effects of temperature and modifier on SFC separations are similar to what has been reported for most other CSPs. We also observed a unique selectivity advantage of SFC for verapamil. We had good success with using a 1-in. diameter column packed with Whelk-O 1 to perform preparative SFC separations of a number of enantiomeric mixtures. The advantages of preparative SFC over preparative HPLC will be discussed. The feasibility of preparative SFC is dependent on how well we meet the practical challenges such as sample introduction issues, special hardware requirements due to the high pressure, and fraction collection issues. © 1994 Wiley-Liss, Inc.     

A practical and reliable method for measuring ethane and pentane in expired air from humans.

We describe a method for the collection of expired air and further document the performance of our analytical technique that is used to measure ethane and pentane simultaneously. Four minutes of breathing hydrocarbon-free air before collection effectively removed high concentrations of residual ambient ethane and pentane from the lungs, with washout times up to 30 min resulting in no further reductions in breath hydrocarbons. Mean (+/-SE) exhalation rates (pmol/kg b.wt./min) in 11 subjects were 2.4 +/- 0.6 for ethane and 1.5 +/- 1.3 for pentane. Total intraindividual variability in exhalation rates (as percent coefficient of variation, %CV), measured from 4 subjects on at least 6 different days, was greater for pentane (44% CV) than for ethane (29% CV). Analytical variability contributed 6% to the total %CV. Advantages of the method are described, and reasons for the large variability in values reported in the literature are discussed.     

大鼠离体心脏停灌和再灌早期引起的肌酸激酶的双相释放

心脏缺血再灌损伤导致肌酸激酶(CK)的大量释放。本实验提供了一个模型、可对再灌早期CK释放的动态变化进行研究。目的在于试图将停灌损伤和再灌损伤加以区分,并探讨氧反常和钙反常在两种损伤中的相对作用。用Langendorff法灌流大鼠离体心脏,平衡10min,停灌10min。于再灌3min内每15s收集一次冠脉流份,测定CK活性(U/L),作为心肌细胞损伤的指标。再灌3min内CK释放呈双相变化,它们的峰值比平衡期对照值高4-6倍。第Ⅰ峰恒定出现于再灌15s。第Ⅱ峰在有基质Krebs-Henseieit(K-H)溶液灌流组主要出现在再灌30-75s,在无基质K-H溶液灌流组主要出现在再灌120-180s。初步判断,第Ⅰ峰主要代表缺灌损伤,第Ⅱ峰主要代表再灌损伤。缺氧或加葡萄糖灌流均能降低双峰值及总释放量,而低钙灌流仅能延缓第Ⅱ峰的出现。由于葡萄糖能增强细胞对氧反常的耐受性,而缺氧能使氧反常推迟出现,又由于缺灌期胞外液并不缺钙,因此在OK双相释放峰值中可能并不包含典型的钙反常成分,而包含氧反常成分。至于低钙降低第Ⅱ峰的原因,可能是暂时抑制了氧反常造成的钙内流与钙负荷损伤。     

Quantitation of sorafenib and its active metabolite sorafenib N-oxide in human plasma by liquid chromatography–tandem mass spectrometry

A simple and rapid method with high performance liquid chromatography/tandem mass spectrometry is described for the quantitation of the kinase inhibitor sorafenib and its active metabolite sorafenib N-oxide in human plasma. A protein precipitation extraction procedure was applied to 50 μL of plasma. Chromatographic separation of the two analytes, and the internal standard [²H₃¹³C]-sorafenib, was achieved on a C₁₈ analytical column and isocratic flow at 0.3 mL/min for 4 min. Mean within-run and between-run precision for all analytes were <6.9% and accuracy was <5.3%. Calibration curves were linear over the concentration range of 50–10,000 ng/mL for sorafenib and 10–2500 ng/mL for sorafenib N-oxide. This method allows a specific, sensitive, and reliable determination of the kinase inhibitor sorafenib and its active metabolite sorafenib N-oxide in human plasma in a single analytical run.     

A rapid protocol for purification of total RNA for tissues collected from pigs at a slaughterhouse

Since RNA extraction is a crucial step in many molecular techniques, the protocols for sample collection and RNA purification need to be adapted to optimize their performance when samples are collected from animals at commercial facilities. Here we provide an RNA purification protocol for animal tissues collected from slaughterhouses. This protocol, modified from other techniques, uses TRIzol Reagent. Sample collection was performed wearing sterile gloves and facemasks, using sterile surgical instruments, and no longer than 8 min spent for each sample. A 0.9% sterile sodium chloride solution was used to wash the tissue before each sample collection. The whole process of RNA extraction was performed under cold environment and sterile conditions. This protocol produced good RNA yields (50 μg RNA per 100 mg tissue), good integrity and purity (Abs(260/280) from 1.8 to 2.0), from tissues such as liver, muscle, hypophysis, adipose tissue, and intestinal mucosa, in less than 2 h.     

Less is more: standard warm-up causes fatigue and less warm-up permits greater cycling power output

The traditional warm-up (WU) used by athletes to prepare for a sprint track cycling event involves a general WU followed by a series of brief sprints lasting ≥ 50 min in total. A WU of this duration and intensity could cause significant fatigue and impair subsequent performance. The purpose of this research was to compare a traditional WU with an experimental WU and examine the consequences of traditional and experimental WU on the 30-s Wingate test and electrically elicited twitch contractions. The traditional WU began with 20 min of cycling with a gradual intensity increase from 60% to 95% of maximal heart rate; then four sprints were performed at 8-min intervals. The experimental WU was shorter with less high-intensity exercise: intensity increased from 60% to 70% of maximal heart rate over 15 min; then just one sprint was performed. The Wingate test was conducted with a 1-min lead-in at 80% of optimal cadence followed by a Wingate test at optimal cadence. Peak active twitch torque was significantly lower after the traditional than experimental WU (86.5 ± 3.3% vs. 94.6 ± 2.4%, P < 0.05) when expressed as percentage of pre-WU amplitude. Wingate test performance was significantly better (P < 0.01) after experimental WU (peak power output = 1,390 ± 80 W, work = 29.1 ± 1.2 kJ) than traditional WU (peak power output = 1,303 ± 89 W, work = 27.7 ± 1.2 kJ). The traditional track cyclist's WU results in significant fatigue, which corresponds with impaired peak power output. A shorter and lower-intensity WU permits a better performance.     

The binary phase behavior of 1,3-dimyristoyl-2-stearoyl-sn-glycerol and 1,2-dimyristoyl-3-stearoyl-sn-glycerol

The binary phase behavior of pure 1,3-dimyristoyl-2-stearoyl-sn-glycerol (MSM) and 1,2-dimyristoyl-3-stearoyl-sn-glycerol (MMS) was investigated in terms of polymorphism, melting and crystallization behavior, SFC, hardness and microstructure. Samples were crystallized at cooling rates of 3.0 and 0.1 degrees C/min. The asymmetric TAG demonstrated lower melting and crystallization points at both cooling rates. All samples crystallized in the beta' polymorph when cooled at 0.1 degrees C/min and in the alpha polymorph when cooled at 3.0 degrees C/min. The experimentally determined kinetic phase diagram of MSM-MMS was monotectic for both cooling rates. This data was well described by a thermodynamic model using the Bragg-Williams approximation for non-ideality of mixing and suggested that in both the solid and liquid states, like pair interactions (MSM-MSM and MMS-MMS) were favored over MSM-MMS interaction. A strong tendency to phase separation in the solid phase was also observed. For both cooling rates, the fit of the SFC (%)-time curves to a modified form of the Avrami model indicated that crystallization occurred in two distinct kinetic steps. Depressions seen in SFC did not correspond to depressions in hardness or melting temperatures.     

Simulated moving columns technique for enantioselective supercritical fluid chromatography

This article describes a very useful extension of an unique column switching technique called "Simulated Moving Columns" (SMC) that was previously reported for chiral high performance liquid chromatography (HPLC) (Zhang and McConnell, Journal of Chromatography A 2004;1028:227-238). SMC uses two or three short chiral columns connected in series, and enables the unresolved enantiomers to separate repeatedly and exclusively through each of the columns until sufficient resolution is attained. The technique is significantly enhanced through the use of supercritical fluid chromatography (SFC). The supercritical or near critical carbon dioxide (CO(2)) used in the mobile phase of SFC possesses the properties of a liquid as well as a gas, and usually results in much sharper peaks compared to HPLC. Consequently, by combining SMC with SFC (SMC-SFC), we were able to dramatically increase the number of SMC cycles with significantly less band broadening compared to HPLC. For the first time, an enantioselective SFC separation was demonstrated by increasing the column from the actual 20 cm length to reach a half meter virtual length with remarkably enhanced efficiency. Off-column band broadening resulting from a two-column SMC system was measured, and its impact on the enantioselectivity of SMC-SFC was found to be much less than in SMC-HPLC.     

除颤时间与心脏性猝死除颤复苏成功率的相关性分析

目的:研究除颤时间与心脏性猝死患者除颤复苏成功率的相关性。方法:选取2015年2月至2017年6月于我院接受除颤复苏治疗的心脏性猝死患者120例为研究对象。分析除颤时间与除颤复苏成功以及心功能舒张早期充盈峰速度(E峰)、左室射血分数(LVEF)、左心室舒张末期内径(LVEDD)以及E/舒张晚期充盈峰速度(A)水平的相关性。结果:电除颤时间2 min患者的复苏成功率为60.00%(21/35),显著高于电除颤时间2~5 min、5~10 min以及10 min患者的34.21%(13/38)、11.11%(3/27)、0.00%(0/20),而电除颤时间2~5 min患者的复苏成功率又显著高于电除颤时间5~10 min患者,差异均有统计学意义(均P0.05)。电除颤时间2 min、2~5 min、5~10 min以及10 min患者的E峰、LVEF、LVEDD以及E/A水平呈逐渐下降趋势,差异均有统计学意义(均P0.05)。Pearson相关性分析结果显示心脏性猝死患者除颤时间与除颤复苏成功率、E峰、LVEF、LVEDD以及E/A均呈负相关关系(r=-0.593,P=0.000;r=-0.476,P=0.001;r=-0.523,P=0.000;r=-0.502,P=0.000;r=-0.469,P=0.001)。结论:除颤时间与心脏性猝死患者除颤复苏成功率呈负相关关系,即除颤时间越早,患者复苏成功率越高。     

A developed determination of midazolam and 1'-hydroxymidazolam in plasma by liquid chromatography-mass spectrometry: application of human pharmacokinetic study for measurement of CYP3A activity

This paper describes sensitive and reliable determination of midazolam (MDZ) and its major metabolite 1'-hydroxymidazolam (1-OHMDZ) in human plasma by liquid chromatography-mass spectrometry (LC-MS) with a sonic spray ionization (SSI) interface. MDZ, 1-OHMDZ and diazepam as an internal standard were extracted from 1ml of alkalinized plasma using n-hexane-chloroform (70:30, v/v). The extract was injected into an analytical column (YMC-Pak Pro C(18), 50mmx2.0mmi.d.). The mobile phase for separation consisted of 10mM ammonium acetate and methanol (50:50, v/v) and was delivered at a flow-rate of 0.2ml/min. The drift voltage was 100V. The sampling aperture was heated at 120 degrees C and the shield temperature was 260 degrees C. The total time for chromatographic separation was less than 16min. The validated concentration ranges of this method were 0.25-50ng/ml for both MDZ and 1-OHMDZ. Mean recoveries were 93.6% for MDZ and 86.6% for 1-OHMDZ. Intra- and inter-day coefficient variations were less than 6.5 and 5.5% for MDZ, and 6.1 and 5.7% for 1-OHMDZ at 0.3, 4, 20 and 40ng/ml. The limits of quantification were 0.25ng/ml for both MDZ and 1-OHMDZ. This method was sensitive and reliable enough for pharmacokinetic studies on healthy volunteers, and was applied for the measurement of CYP3A activity in humans after an intravenous (1mg) and a single-oral administration (2mg) of subtherapeutic MDZ dose.     

Effect of system variables involved in packed column SFC of nevirapine as model analyte using response surface methodology: application to retention thermodynamics, solute transfer kinetic study and binary diffusion coefficient determination

A multifactor optimization technique is successfully applied to study the effect of simultaneously varying the system variables on feasibility of nevirapine analysis by packed column supercritical fluid chromatography (PC-SFC). The optimal conditions were determined with the aid of the response surface methodology using 3(3) factorial designs. The method is based on methanol-modified carbon dioxide as the mobile phase at flow rate of 3.0 ml/min with elution through a JASCO Finepak SIL-5, [C18 (5-micron, 25 cm x 4.6 mm, i.d.)] column using photodiode array detection. The method has been successfully used to analyze commercial solid dosage form to assess the chromatographic performance of SFC system. The present work briefs the thermodynamic applications of PC-SFC with an emphasis on the results of nevirapine. The foremost of such applications is the determination of solute diffusion coefficient in supercritical mobile phase by Taylor-Aris peak broadening technique.     

Determination of a novel gamma-secretase inhibitor in human plasma and cerebrospinal fluid using automated 96 well solid phase extraction and liquid chromatography/tandem mass spectrometry

A method for determination of a gamma-secretase inhibitor, cis-3-[4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]propanoic acid (A), in human plasma and cerebrospinal fluid (CSF) has been developed to support the clinical investigation of compound A for its potential treatment of Alzheimer's disease. The method is based on HPLC with atmospheric pressure chemical ionization tandem mass spectrometric detection (APCI-MS/MS) in the negative ionization mode using a heated nebulizer interface. The addition of phosphoric acid at the ratio of 10-30microL per milliliter of human plasma or CSF was required during clinical sample collection to stabilize an acylglucuronide metabolite (C), which was potentially present in human plasma and CSF. Tween 20 (10% solution) was added at the ratio of 20microL per milliliter of CSF during CSF sample collection to prevent the loss of compound A during the storage of clinical samples. The compound A and its analog internal standard (B) in treated plasma or CSF were isolated from human plasma or CSF using solid phase extraction (SPE) in the 96 well format. The isolated analyte and internal standard were chromatographed on a Phenomenex Synergi Polar RP analytical column (50mmx3.0mm, 4microm), using a mobile phase consisting of 60/40 (v/v, %) acetonitrile/water at a flow-rate of 0.5mL/min. Tandem mass spectrometric detection was performed using a Sciex API 3000 tandem mass spectrometer operated in the multiple reaction monitoring (MRM) mode using precursor to product ion transitions of 441-->175 for A and 469-->175 for B, respectively. The assays were validated over the concentration range of 0.5-200ng/mL for human plasma and CSF. Replicate analyses (n=5) of spiked standards for both assays yielded a linear response with coefficients of variation less than 7% and accuracy within 5% of the nominal concentrations. In addition, the assays were automated to improve sample throughput by utilizing a Packard Multi PROBEII automated liquid handling system and a Tom-Tec Quadra 96 system. Numerous clinical studies have been supported using these assays.     

Hypothalamo-pituitary portal blood concentrations of beta-endorphin during suckling in the ewe

Matched hypothalamo-pituitary portal and jugular blood samples were collected over about 6 h from 7 lactating Corriedale ewes penned with their lambs, and a careful record was kept of ewe/lamb behaviour. Hypothalamo-pituitary portal blood concentrations of beta-endorphin were measured by radioimmunoassay and the secretion rates were calculated; these were related to peripheral plasma prolactin and LH concentrations, and the sucking bouts of the lambs. Basal LH concentrations remained less than 1 ng/ml with 0-2 pulses of 1.5-3.5 ng/ml amplitude per 6-h collection period. Prolactin secretion was episodic with individual baselines varying from 24 to 286 ng/ml, and peak concentrations of 50-631 ng/ml. Portal beta-endorphin was secreted in an episodic pattern with individual baseline secretion rates varying from 0.125 to 0.495 ng/min, and peak secretion rates of 0.768 to 3.216 ng/min. A close correlation was seen between sucking bouts and the secretion of portal beta-endorphin and peripheral prolactin; 86% of sucking bouts resulted in a significant release of beta-endorphin, and 46% of sucking bouts resulted in a significant release of prolactin. These results show that hypothalamic beta-endorphin is released in response to the sucking stimulus. This provides support for the hypothesis that, during lactation, beta-endorphin acts within the hypothalamus to reduce GnRH release and hence depress pituitary gonadotrophin secretion.     

Development and validation of a selective and robust LC-MS/MS method for high-throughput quantifying rizatriptan in small plasma samples: application to a clinical pharmacokinetic study

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection (LC-MS/MS) was developed for the determination of a potent 5-HT(1B/1D) receptor agonist, rizatriptan in human plasma using granisetron as the internal standard. The analyte and internal standard were isolated from 100 microL plasma samples by liquid-liquid extraction (LLE) and chromatographed on a Lichrospher C18 column (4.6mm x 50mm, 5 microm) with a mobile phase consisting of acetonitrile-10mM aqueous ammonium acetate-acetic acid (50:50:0.5, v/v/v) pumped at 1.0 mL/min. The method had a chromatographic total run time of 2 min. A Varian 1200 L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 270-->201 (rizatriptan) and 313.4-->138 (granisetron) used for quantitation. The assay was validated over the concentration range of 0.05-50 ng/mL and was found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction recovery from spiked plasma samples was above 98%. The intra-day accuracy of the assay was within 12% of nominal and intra-day precision was better than 13% C.V. Following a 10mg dose of the compound administered to human subjects, mean concentrations of rizatriptan ranged from 0.2 to 70.6 ng/mL in plasma samples collected up to 24h after dosing. Inter-day accuracy and precision results for quality control samples run over a 5-day period alongside clinical samples showed mean accuracies of within 12% of nominal and precision better than 9.5% C.V.     

Effects of rapid changes in oxygen concentration on the respiration of carrot roots

Rates of CO₂ production and O₂ consumption from aged disks of carrot ( Daucus carota L.) root tissues were measured for 4 h after they were transferred from 21% to 0, 1, 2, 4 or 8% O₂ in gas mixtures. A transient peak in the rate of CO₂ production started 5 to 7 min after transfer to 2% or lower O₂ mixtures and peaked at 50 min. After the peaks in CO₂ production from the 0, 1 and 2% O₂ treatments and after the stable production from the 4 and 8% O₂ treatments, the rate of CO₂ production from all low O₂ treatments started to decline at 50 min, reaching stable rates by 160 to 240 min. Concentrations of lactate and ethanol that were significantly higher than the 21% O₂ controls had started to accumulate in disks between 10 and 50 min after exposure to atmospheres containing 2% or less O₂. Production of CO₂ started to increase 5 to 7 min after transfer to 0, 1 and 2% O₂, while the initial decline and then rise in pH and the accumulation of ethanol did not occur until 30 min after the change in atmosphere. Ethanol accumulation paralleled the increase in pH; first at 0.4 μmol g⁻¹ h⁻¹ from 30 to 60 min as the pH shifted from 5.97 to 6.11, and then at 0.08 μmol g⁻¹ h⁻¹ from 60 to 100 min as the pH stablized around 6.12. The peak at 50 min in CO₂ production roughly coincided with the shift from the rapid to the slow change in pH and ethanol accumulation.     

Separation and trace estimation of benzidine and its macromolecular adducts using supercritical fluid chromatography

A sensitive, rapid, selective and reproducible method has been developed to measure blood plasma levels of benzidine (BZ) and its acetylated metabolite, N-OH-N,N'-diacetylbenzidine (N-OH-DABZ), using supercritical fluid chromatography (SFC) for the first time. Benzidine and N-OH-N,N'-diacetylbenzidine were extracted from the plasma using ether. Separation was done on a Nucleosil (250 mm x 4.6 mm) 10 microm, Nucleosil-RP-C18 column with 7.4% (v/v) methanol-modified supercritical fluid carbon dioxide (2.5 ml min(-1)) as mobile phase. The column temperature was 45 degrees C and the outlet pressure was set at 8.83 MPa. The detection was done using a UV-Vis detector set at 280 nm. The limit of quantification was 0.10 ng ml(-1) (BZ) and 0.14 ng ml(-1) (N-OH-diacetylbenzidine) using 1 ml plasma specimen. The mean extraction recovery of BZ was found to be 98.6%. The SFC method was directly compared to a published HPLC-UV method. With respect to speed, organic solvent usage, sensitivity, specificity and accuracy, SFC was found to be superior. The method has been successfully used to estimate the BZ, N-OH-diacetylbenzidine levels in blood plasma of the animals who were administered 15 microg kg(-1) body weight of benzidine.Further, this method has been also applied for the detection and quantification of benzidine DNA and hemoglobin adducts from the blood and tissue samples of the benzidine dosed animals.     

Seminal collection, seminal characteristics and pattern of ejaculation in llamas

Semen was collected from 10/10 llamas during 26/30 (87%) collection attempts using an artificial vagina mounted inside a surrogate female. For the 26 semen collections, the duration of copulation (mount to dismount) with the artificial vagina was 31.7 +/- 12.0 min (mean +/- SD). Seminal pH was 8.1 +/- 1.1, and seminal volume per collection was 3.0 +/- 1.9 ml. Sperm concentration per collection was 1.0 +/- 0.8 x 10(6) sperm/ml, total number of spermatozoa was 2.9 +/- 3.1 x 10(6), total sperm motility was 23.7 +/- 20.0%, and the percentage of morphologically normal spermatozoa was 39.7 +/- 18.5%. Morphologically abnormal spermatozoa were categorized according to abnormal heads (20.1 +/- 19.9%), tail-less heads (8.7 +/- 8.9%), abnormal acrosomes (12.9 +/- 12.4%), abnormal midpieces (1.0 +/- 3.7%), cytoplasmic droplets (11.1 +/- 12.4%), and abnormal tails (6.6 +/- 12.0%). There were 0.3 +/- 0.3 million motile, morphologically normal spermatozoa per collection: less than 1000 during the first 5 min of copulation, 0.01 +/- 0.01 x 10(6) between 5 and 10 min of copulation, 0.04 +/- 0.08 x 10(6) between 10 and 15 min of copulation, 0.09 +/- 0.21 x 10(6) between 15 and 20 min of copulation, and 0.15 +/- 0.28 x 10(6) between 20 min and the end of copulation.     

Quantitative determination of trans-polydatin, a natural strong anti-oxidative compound, in rat plasma and cellular environment of a human colon adenocarcinoma cell line for pharmacokinetic studies

A simple, accurate, precise, specific and reproducible high-performance liquid chromatography (HPLC) method was developed for determination of trans-polydatin, a natural strong anti-oxidative compound, in rat plasma and cell suspension. The assay procedure involved simple liquid-liquid extraction, the supernatant liquid was added an equal volume of water to avoid solvent effect. The detection of the analyte peak was achieved by monitoring the eluate using a UV detector set at 303 nm. The analysis used a Hypersil ODS2 C18 column (5 microm, 4.6 mm x 250 mm) and methanol/distilled water as the mobile phase (flow rate=1 mL/min). A total analytical run was achieved within 6.0 min and calibration curve was linear over a wide concentration range of 0.25-40 microg/mL for plasma sample and 1.0-500 microM for cell suspension, the coefficients of correlation were 0.9997 and 0.9999 or better, respectively. There was 80.7+/-7.86%, 96.8+/-3.20% and 102.7+/-9.72% recovery from 0.5, 10, and 40 microg/mL plasma samples, respectively. Intra- and inter-batch accuracy and precision were acceptable for the both matrices. The RSD of intra- and inter-day assay variations were all less than 10%. Both analyte and IS were stable in the battery of stability studies, freeze-thaw cycles. The described assay method was applied to pharmacokinetic studies in rats and a human colon adenocarcinoma cell line (Caco-2) successfully. The application of the assay to determine the pharmacokinetic is described.