Cigarette smokers differ in their handling of natural (RRR) and synthetic (all rac) alpha-tocopherol: a biokinetic study in apoE4 male subjects

We have compared the biokinetics of deuterated natural (RRR) and synthetic (all rac) alpha-tocopherol in male apoE4-carrying smokers and nonsmokers. In a randomized, crossover study subjects underwent two 4-week treatments (400 mg/day) with undeuterated RRR- and all rac-alpha-tocopheryl acetate around a 12-week washout. Before and after each supplementation period subjects underwent a biokinetic protocol (48 h) with 150 mg deuterated RRR- or all rac-alpha-tocopheryl acetate. During the biokinetic protocols, the elimination of endogenous plasma alpha-tocopherol was significantly faster in smokers (P < 0.05). However, smokers had a lower uptake of deuterated RRR than nonsmokers, but there was no difference in uptake of deuterated all rac. The supplementation regimes significantly raised plasma alpha-tocopherol (P < 0.001) with no differences in response between smokers and nonsmokers or between alpha-tocopherol forms. Smokers had significantly lower excretion of alpha-carboxyethyl-hydroxychroman than nonsmokers following supplementation (P < 0.05). Nonsmokers excreted more alpha-carboxyethyl-hydroxychroman following RRR than all rac; however, smokers did not differ in excretion between forms. At baseline, smokers had significantly lower ascorbate (P < 0.01) and higher F(2)-isoprostanes (P < 0.05). F(2)-isoprostanes in smokers remained unchanged during the study, but increased in nonsmokers following alpha-tocopherol supplementation. These data suggest that apoE4-carrying smokers and nonsmokers differ in their handling of natural and synthetic alpha-tocopherol.     

Comparison of gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry methods to quantify alpha-tocopherol and alpha-tocopherolquinone levels in human plasma.

Two mass spectrometric methods were established for the quantitative analyses of alpha-tocopherol (TH) and its oxidation product alpha-tocopherolquinone (TQ) in human plasma. Both methods make use of isotopically labeled internal standards of different levels of deuteration (d3-TH and d6-TQ). Plasma (100 microl) was saponified in the presence of a mixture of antioxidants, and then TH and TQ were extracted with hexane. With the GC-MS method, the analytes were first converted into O-trimethylsilyl derivatives before analysis in the selective ion monitoring mode. The derivatization procedure led to the quantitative conversion of TQ into the O-trimethylsilyl derivative of tocopherolhydroquinone, giving rise to a more stable molecule with less fragmentation than for TQ. The increased stability of the molecule resulted in an enhanced contribution of the base peak to the total observed ions and therefore an increased sensitivity of the base peak for quantification. With the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, TH and TQ were detected by multiple reaction monitoring after positive electrospray ionization. The GC-MS and LC-MS/MS methods showed nearly the same accuracy (>95%) and the same within-day precisions, with less than 5 and 10% for TH and TQ, respectively. The between-day precision and the limit of quantification for TQ in plasma were better by LC-MS/MS (4%; 3 nM) than by GC-MS (21%; 10 nM). Analysis and method validation were carried out with plasma samples obtained from a male volunteer pre- and postexercise. Both techniques showed that the ratio of TQ/TH was elevated by 35% immediately after exercise and had returned to basal levels when measured 24 h later.     

Vitamin E bioavailability in humans

It is important to understand factors that can influence vitamin E bioavailability, particularly in populations with increased risk of coronary heart disease such as cigarette smokers. There is also a need to clarify the bioavailability of natural and synthetic vitamin E, which is currently a subject of some controversy. Previous studies using a competitive uptake approach have found bioavailability ratios of natural:synthetic vitamin E close to 2:1, differing from the accepted biopotency ratio of 1.36:1. We used a non-competitive uptake approach to compare the plasma biokinetics of deuterated natural (RRR) and synthetic (all rac) alpha-tocopheryl acetate in smokers and non-smokers. The study was comprised of two 4-week treatments with 400 mg/d (either RRR-alpha-tocopheryl or all rac-alpha-tocopheryl acetates), with a 12-week washout period between. Prior to and after each treatment subjects underwent a 48 h biokinetic protocol with 150 mg deuterated alpha-tocopheryl acetate in either the RRR or all rac form. Smokers had a lower deuterated alpha-tocopherol AUC than did non-smokers following administration of RRR, but there was no difference following administration of all rac. The ratio RRR:all rac from AUCs and C(max) was 1.3:1 in non-smokers and 0.9:1 in smokers. Following vitamin E supplementation, deuterated tocopherol AUCs were lower in both groups. These data suggest that non-smokers and smokers differ in their handling of vitamin E, that the relative bioavailability of natural and synthetic vitamin E is close to the currently accepted biopotency ratio of 1.36:1, and that following supplementation the ability of the plasma to take up newly absorbed vitamin E is decreased.     

Quantitative analysis of acrylamide labeled serum proteins by LC-MS/MS

Isotopic labeling of cysteine residues with acrylamide was previously utilized for relative quantitation of proteins by MALDI-TOF. Here, we explored and compared the application of deuterated and (13)C isotopes of acrylamide for quantitative proteomic analysis using LC-MS/MS and high-resolution FTICR mass spectrometry. The method was applied to human serum samples that were immunodepleted of abundant proteins. Our results show reliable quantitation of proteins across an abundance range that spans 5 orders of magnitude based on ion intensities and known protein concentration in plasma. The use of (13)C isotope of acrylamide had a slightly greater advantage relative to deuterated acrylamide, because of shifts in elution of deuterated acrylamide relative to its corresponding nondeuterated compound by reversed-phase chromatography. Overall, the use of acrylamide for differentially labeling intact proteins in complex mixtures, in combination with LC-MS/MS provides a robust method for quantitative analysis of complex proteomes.     

Quantitative analysis of [Dmt(1)]DALDA in ovine plasma by capillary liquid chromatography-nanospray ion-trap mass spectrometry

The synthetic opioid peptide analog Dmt-D-Arg-Phe-Lys-NH(2) ([Dmt(1)]DALDA; [Dmt= 2',6'-dimethyltyrosine) is a highly potent and selective mu opioid-receptor agonist. A very sensitive and robust capillary liquid chromatography/nanospray ion-trap (IT) mass spectrometry method has been developed to quantify [Dmt(1)]DALDA in ovine plasma, using deuterated [Dmt(1)]DALDA as the internal standard. The standard MS/MS spectra of d(0)- and d(5)-[Dmt(1)]DALDA were obtained, and the collision energy was experimentally optimized to 25%. The product ion [ M + 2H-NH(3)](2+) (m/z 312.2) was used to identify and to quantify the synthetic opioid peptide analog in ovine plasma samples. The MS/MS detection sensitivity for [Dmt(1)]DALDA was 625 amol. A calibration curve was constructed, and quantitative analysis was performed on a series of ovine plasma samples.     

Vitamin E kinetics in smokers and nonsmokers.

Does cigarette smoking increase vitamin E utilization in vivo? A trial was carried out in 6 smokers and 5 nonsmokers of comparable ages and serum lipids. Subjects consumed 75 mg each d(3)-RRR and d(6)-all rac-alpha-tocopheryl acetates (natural and synthetic vitamin E, respectively) daily for 7 d with a standardized breakfast. Fasting blood samples were drawn on days -7, -6, -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 9, 14, 21 (negative days indicate supplementation). In both groups, plasma d(3)-alpha-tocopherol concentrations were approximately double of d(6)-alpha-tocopherol. At day 0, the %d(3) alpha-tocopherols (d(3)-alpha-tocopherol/total-alpha-tocopherol x 100) were similar in both smokers and nonsmokers. Subsequently, there was a trend toward a faster exponential disappearance of the plasma %d(3) alpha-tocopherol in smokers compared with nonsmokers (0.30 +/- 0.04 compared with 0.24 +/- 0.05, p =.0565). The calculated %d(3) half-lives were 55.6 +/- 7.4 h in smokers and 72.1 +/- 17.3 h in nonsmokers (p =.0630). By day 21, the %d(3) in smokers had decreased to 1.4% +/- 0.3% while it was 2.2% +/- 0.7% (p =.0418) in the nonsmokers. These data suggest that smoking increases plasma vitamin E disappearance, but further studies are needed to confirm this finding and to assess its cause.     

Quantitation of rat liver vitamin E metabolites by LC-MS during high-dose vitamin E administration

To evaluate vitamin E metabolism, a method was developed to quantitate liver alpha- and gamma-tocopherol metabolites, alpha-carboxyethyl hydroxychroman [alpha-CEHC; 2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman] and gamma-CEHC [2,7,8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman], respectively. Vitamin E supraenriched livers were obtained from rats that were injected with vitamin E daily for 18 days. Liver samples (approximately 50 mg) were homogenized, homogenate CEHC-conjugates were hydrolyzed, CEHCs were extracted with ethyl ether, and then CEHCs were quantitated using liquid chromatography-mass spectrometry (LC-MS). Precision, based on intersample variability, ranged from 1% to 3%. Recovery of alpha- and gamma-CEHCs added to liver homogenates ranged from 77% to 87%. Detection limits of alpha- and gamma-CEHC were 20 fmol, with a linear detector response from 0.025 to 20 pmol injected. Corresponding with an increase in liver alpha-tocopherol, the MS peak for liver alpha-CEHC (mass-to-charge ratio 277.8) increased 80-fold (0.18 +/- 0.01 to 15 +/- 2 nmol/g). Liver alpha-CEHC concentrations were correlated with serum alpha-CEHC, liver alpha-tocopherol, and serum alpha-tocopherol (P < 0.001 for each comparison). alpha-CEHC represented 0.5-1% of the liver alpha-tocopherol concentration. Thus, LC-MS can be successfully used to quantitate alpha- and gamma-CEHC in liver samples. These data suggest that in times of excess liver alpha-tocopherol, increased metabolism of alpha-tocopherol to alpha-CEHC occurs.     

Easy preparation of a large-size random gene mutagenesis library in Escherichia coli

Osmolytes are accumulated intracellularly to offset the effects of osmotic stress and protect cellular proteins against denaturation. Because different taxa accumulate different osmolytes, they can also be used as "dietary biomarkers" to study foraging. Potential osmolyte biomarkers include glycine betaine, trimethylamine N-oxide (TMAO), homarine, dimethylsulfoniopropionate (DMSP), and the osmolyte analog arsenobetaine (AsB). We present a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the simultaneous measurement of these osmolytes in serum or plasma. Varying concentrations of osmolytes were added to serum and samples and extracted in 90% acetonitrile and 10% methanol containing 10 μM deuterated internal standards (D(9)-glycine betaine, D(9)-trimethylamine-N-oxide, (13)C(2)-arsenobetaine, D(6)-DMSP, and D(4)-homarine). Analytes were separated on a normal-phase modified silica column and detected using isotope dilution tandem mass spectrometry in multiple reaction monitoring (MRM) mode. The assay was linear for all six compounds (r(2) values=0.983-0.996). Recoveries were greater than 85%, and precision for within-batch coefficients of variation (CVs) were less than 8.2% and between-batch CVs were less than 6.1%. Limits of detection ranged from 0.02 to 0.12 μmol/L. LC-MS/MS is a simple method with high throughput for measuring low levels of osmolytes that are often present in biological samples.     

Studies in humans using deuterium-labeled alpha- and gamma-tocopherols demonstrate faster plasma gamma-tocopherol disappearance and greater gamma-metabolite production

We hypothesized that human plasma alpha- and gamma-tocopherol concentrations reflect differences in their kinetics, especially influenced by gamma-tocopherol metabolism. Vitamin E kinetics were evaluated in humans (n=14) using approximately 50 mg each of an equimolar ratio of d6-alpha- and d2-gamma-tocopheryl acetates administered orally. Mass spectrometry was used to measure deuterated plasma tocopherols, as well as plasma and urinary vitamin E metabolites, alpha- and gamma-carboxyethylhydroxychromans (CEHCs). Plasma d2-gamma-tocopherol fractional disappearance rates (FDR; 1.39+/-0.44 pools/day, mean+/-SD) were more than three times greater than those of d6-alpha-tocopherol (0.33+/-0.11, p<0.001). The d2-gamma-tocopherol half-life was 13+/-4 h compared with 57+/-19 for d6-alpha-tocopherol. Whereas neither plasma nor urinary d6-alpha-CEHC was detectable (limit of detection 1 nmol/L), gamma-CEHC (labeled plus unlabeled) increased from 129+/-20 to 258+/-40 nmol/L by 12 h and returned to baseline by 48 h; at 12 h d2-gamma-CEHC represented 54+/-4% of plasma gamma-CEHC. Women compared with men had a greater d2-gamma-tocopherol FDR (p<0.004) and a greater maximal plasma d2-gamma-CEHC concentration (p<0.02) and CEHC FDR (p<0.007), as well as excreting four times as much d2-gamma-CEHC (p<0.04) in urine. Thus, gamma-tocopherol is rapidly metabolized to gamma-CEHC, and to a greater degree in women than in men, whereas alpha-tocopherol is maintained in the plasma and little is metabolized to alpha-CEHC.     

Liquid chromatography-tandem mass spectrometry assay for the quantitation of beta-dihydroartemisinin in rat plasma

Dihydroartemisinin (DHA) is a sesquiterpene used in the world as an antimalarial. To evaluate the pharmacokinetics of dihydroartemisinin in rats, a sensitive and specific liquid chromatography/tandem mass spectrometric (LC-MS/MS) method was developed and validated for the quantitation of dihydroartemisinin in rat plasma. For detection, a Sciex API 4000 LC-MS/MS with a TurboIonSpray ionization (ESI) inlet in the positive ion-multiple reaction monitoring (MRM) mode was used. The plasma samples were pre-treated by a simple liquid-liquid extraction with diethyl ether. The statistical evaluation for this method reveals excellent linearity, accuracy and precision for the range of concentrations 0.2-100.0 ng/mL. The method had a lower limit of quantification (LLOQ) of 0.2 ng/mL for beta-dihydroartemisinin in 100 microL of plasma. The method was successfully applied to the characterization of the pharmacokinetic profile of beta-dihydroartemisinin in rats after oral administration.     

A rapid and sensitive method for the quantitation of montelukast in sheep plasma using liquid chromatography/tandem mass spectrometry

A rapid LC-MS/MS method was developed and partially validated for the quantitation of montelukast in spiked sheep plasma. A total run time of 1.5 min was achieved using a short monolithic column and employing a rapid gradient. Sample preparation involved protein precipitation with twofold acetonitrile by volume during which a deuterated internal standard (montelukast D-6) was incorporated. The MRM transitions for montelukast and the deuterated internal standard were 586/422 and 592/427, respectively. A linear dynamic range of 0.25-500 ng/mL with a correlation coefficient of 0.9999 was achieved. Precision was below 5% at all levels except at the LOQ (0.36 ng/mL) which demonstrated an overall of R.S.D. of 8%. Post-column infusion experiments were performed with precipitated plasma matrix and showed minimal interference with the peaks of interest.     

Liquid chromatography-tandem mass spectrometric quantification of the dehydration product of tetranor PGE-M, the major urinary metabolite of prostaglandin E(2) in human urine

A LC-MS/MS method has been developed to analyze tetranor PGE-M, the major urinary metabolite of PGE(2), that involves the acid-catalyzed dehydration of tetranor PGE-M and its deuterated (d(6)) analog followed by LC-MS/MS measurement of the dehydrated tetranor PGE-M product (tetranor PGA-M). We also report a method for quantification of creatinine in urine by LC-MS/MS to normalize tetranor PGE-M concentrations with that of urinary creatinine. These methods were used to study the effect of aspirin on urinary tetranor PGE-M levels in healthy male volunteers. Aspirin did not affect urinary creatinine concentrations but decreased urinary tetranor PGE-M concentrations by approximately 44%.     

Nascent VLDL from liver perfusions of cynomolgus monkeys are preferentially enriched in RRR- compared with SRR-alpha-tocopherol: studies using deuterated tocopherols

The transport and secretion of vitamin E in lipoproteins have been studied in cynomolgus monkeys fed tocopherols labeled with different amounts of deuterium. The animals were fed a single dose of vitamin E containing 60 mumol of each 2R,4'R,8'R-alpha-(5,7-(C2H3)2)tocopheryl acetate (d6-RRR-alpha-tocopheryl acetate; alpha-tocopherol with natural stereochemistry), 2S,4'R,8'R-alpha-5-(C2H3)tocopheryl acetate (d3-SRR-alpha-tocopheryl acetate; alpha-tocopherol with unnatural stereochemistry), and 2R,4'R,8'R-gamma-(3,4-2H)tocopherol (d2-RRR-gamma-tocopherol; gamma-tocopherol with natural stereochemistry). Chylomicrons, as well as the other plasma lipoproteins, contained equal concentrations of all three tocopherols at the earliest time points after feeding suggesting that all three tocopherols were absorbed equally. At later times plasma lipoproteins became preferentially enriched in d6-RRR-alpha-tocopherol. This is likely to be due to hepatic secretion of VLDL (very low density lipoproteins) and other lipoproteins, which were enriched in d6-RRR-alpha-tocopherol, as demonstrated in the lipoproteins isolated from perfused livers that had been obtained 24 h following the administration of the deuterated tocopherols. Taken together these data demonstrate that the liver, not the intestine, is the likely site of discrimination between tocopherol isomers and that the liver secretes nascent lipoproteins preferentially enriched in d6-RRR-alpha-tocopherol.     

Vitamin E delivery to human skin: studies using deuterated alpha-tocopherol measured by APCI LC-MS

Enrichment of skin surface lipids with deuterium-labeled alpha-tocopherol was compared with plasma enrichment to evaluate kinetics of the delivery of vitamin E to skin surface lipids. For 7 d, subjects consumed 75 mg each of RRR-alpha-[5-(C2H3)]- (d3) and all rac-alpha-[5,7-(C2H3)2]- (d6) tocopheryl acetates with breakfast. Blood was drawn and skin lipids were collected daily for 2 weeks, then every other day for 2 weeks. A liquid chromatography-mass spectrometry atmospheric pressure chemical ionization method for quantification of deuterium labeled (d3, d6, d9-alpha-tocopherols) and unlabeled (d0-) alpha- and gamma-tocopherols was developed. Tocopherols were quantified at their m/z [M-1] using single ion recording. alpha-Tocopherol detection was linear from 1 to 100 pmol with a detection limit of 40 pg (93 fmol). Detection of gamma-tocopherol was twice as sensitive due to greater ionization efficiency. Though d3- and d6-alpha-tocopherols appeared in plasma within 24 h of the first dose, d3-alpha-tocopherol was not detected in skin surface lipids until approximately 1 week. Plasma percentage d3 peaked at day 8, while skin surface lipid percentage d3 increased on average until day 19. Apparently skin employs a mechanism to deliver alpha-tocopherol into skin via lipid secretions.     

Liquid Chromatography-Tandem Mass Spectrometry for Analysis of Intestinal Permeability of Loperamide in Physiological Buffer

Analysis of in vitro samples with high salt concentrations represents a major challenge for fast and specific quantification with liquid chromatography-tandem mass spectrometry (LC-MS/MS). To investigate the intestinal permeability of opioids in vitro employing the Ussing chamber technique, we developed and validated a fast, sensitive and selective method based on LC–MS/MS for the determination of loperamide in HEPES-buffered Ringer''s solution. Chromatographic separation was achieved with an Atlantis dC18 column, 2.1 mm×20 mm, 3 µm particle size and a gradient consisting of methanol/0.1% formic acid and ammonium acetate. The flow rate was 0.7 ml/min, and the total run time was 3 min. For quantification, two mass transitions for loperamide and a deuterated internal standard (methadone-d₃) were used. The lower limit of loperamide quantification was 0.2 ng/ml. This new LC-MS/MS method can be used for the detection of loperamide in any experimental setup using HEPES-buffered Ringer''s solution as a matrix compound.     

Liquid chromatography/negative ion electrospray tandem mass spectrometry method for the quantification of rosuvastatin in human plasma: application to a pharmacokinetic study

A sensitive liquid chromatography/tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of rosuvastatin in human plasma. The plasma samples were prepared using liquid-liquid extraction with ethyl ether. Chromatographic separation was accomplished on a Zorbax XDB-C18 (150 mm x 4.6 mm i.d., 5 microm) column. The mobile phase consisted of methanol-water (75:25, v/v, adjusted to pH 6 by aqueous ammonia). Detection of rosuvastatin and the internal standard (IS) hydrochlorothiazide was achieved by ESI MS/MS in the negative ion mode. The lower limit of quantification was 0.020 ng/ml by using 200 microl aliquots of plasma. The linear range of the method was from 0.020 to 60.0 ng/ml. The intra- and inter-day precisions were lower than 8.5% in terms of relative standard deviation (RSD), and the accuracy was within -0.3 to 1.9% in terms of relative error (RE). Compared with the existing methods, the validated method offered increased sensitivity. The method was successfully applied for the evaluation of pharmacokinetics of rosuvastatin after single oral doses of 5, 10 and 20 mg rosuvastatin to 10 healthy volunteers.     

Rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of clonidine in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the estimation of clonidine in human plasma. Clonidine was extracted from human plasma by using solid-phase extraction technique. Nizatidine was used as the internal standard. A Hypurity C18 (50 mm x 4.6 mm i.d., 5 microm particle size) column provided chromatographic separation of analyte followed by detection with mass spectrometry. The method involves a rapid solid-phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection up to picogram levels with a total run time of 3.0 min only. The method was validated over the range of 50-2500 pg/mL. The absolute recoveries for clonidine (71.86%) and IS (69.44%) achieved from spiked plasma samples were consistent and reproducible.     

Determination of HS270, a new histone deacetylase inhibitor, in rat plasma by LC-MS/MS--application to a preclinical pharmacokinetic study

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and fully validated to determine HS270, a new histone deacetylase (HDAC) inhibitor, in rat plasma using SAHA as the internal standard (IS). After a single step liquid-liquid extraction with acetoacetate, analytes were subjected to LC-MS/MS analysis using positive electro-spray ionization (ESI(+)) under selected reaction monitoring mode (SRM). The chromatographic separation was achieved on a Hypurity C(18) column (50 mm × 2.1 mm, i.d., 5 μm). The MS/MS detection was conducted by monitoring the fragmentation of m/z 392.3→100.1 for HS270, m/z 265.1→232.1 for IS. The method had a chromatographic running time of 2.5 min and linear calibration curves over the concentrations of 0.5-1000 ng/mL. The recovery of the method was 70.8-82.5% and the lower limit of quanti?cation (LLOQ) was 0.5 ng/mL. The intra- and inter-batch precisions were less than 15% for all quality control samples at concentrations of 1.0, 100.0, and 750.0 ng/mL. The validated LC-MS/MS method has successfully applied to a HS270 pharmacokinetic study after oral doses of 25, 50, 100, 200 mg/kg, and i.v. dose of 5 mg/kg to rats.     

Inter- and intra-individual variation in plasma and red blood cell vitamin E after supplementation

To establish the range of individual blood responses to supplemental vitamin E, 30 healthy subjects ingested 75 mg of deuterium-labelled alpha-tocopherol with a standard breakfast. Blood was collected at 6, 9, 12, 27 and 51 h post ingestion and deuterated (d6) and non-deuterated (do) alpha-tocopherol concentrations were determined in plasma and red blood cells (RBC) by GC-MS. To examine intra-individual responses, 6 of these subjects were re-examined at 6-month intervals over a 30-month period. Post ingestion, the amount of d6-alpha-tocopherol in blood increased rapidly with time with maximal concentrations seen at 12 h (plasma) and 27 h (RBC) in most subjects. At these times, d6-alpha-tocopherol concentration ranged from 0.3-12.4 micromol/l in plasma and 0.6-4.09 micromol/l packed cell in RBC. Area under the curve calculations indicated inter-individual differences of alpha-tocopherol uptake to be 40-fold for plasma (12.9-493.3 micromol h/l) and 6-fold for RBC (24.4-146.1 micromol h/l packed RBC). Intra-individual variation in alpha-tocopherol uptake was small in comparison and remained relatively constant over the 30-month period. We conclude that vitamin E uptake varies widely in the normal population, although it is comparatively stable for an individual over time. These differences likely arise from variations in the regulation of vitamin E uptake and metabolism between subjects. Factors regulating this process must be better understood before the optimal intake of vitamin E can be ascertained.     

Simultaneous determination of berberine, palmatine and jatrorrhizine by liquid chromatography-tandem mass spectrometry in rat plasma and its application in a pharmacokinetic study after oral administration of coptis-evodia herb couple

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to simultaneously determine berberine, palmatine and jatrorrhizine in rat plasma. After mixing with the internal standard (IS) tetrahydropalmatine, plasma samples were pretreated by protein precipitation with acetonitrile-methanol (1:2, v/v). Chromatographic separation was carried out on a C18 column using a mixture of water (containing 0.1% formic acid) and acetonitrile (30:70, v/v) as mobile phase. The detection was performed by selected reaction monitoring (SRM) mode via electrospray ionization (ESI) source operating in the positive ionization mode. The method was linear over the concentration range of 1.0-250.0 ng/mL for all components. The intra- and inter-day precision values were less than 14.6% and the deviations were within +/-4.0%. The fully validated LC-MS/MS method has been successfully applied to the pharmacokinetic study of berberine, palmatine and jatrorrhizine in rat plasma after oral administration of coptis-evodia herb couple. Three peaks were observed in both individual and mean plasma-concentration curves of berberine, palmatine and jatrorrhizine, which may be attributed to distribution re-absorption and enterohepatic circulation.