A novel multi-array immunoassay device for tumor markers based on insert-plug model of piezoelectric immunosensor

A novel multi-array immunoassay device based on the insert-plug model of piezoelectric (Pz) immunosensor fabricated with the screw clamp apparatus has been developed for quantitative detection of tumor markers such as alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), prostate specific antigen (PSA), and carcinoma antigen 125 (CA125) in serum, in which single immunosensor can oscillate independently with the frequency stability of +/-1 Hz (hertz) in air phase and +/-2 Hz in liquid phase. These response characteristics of Pz tumor marker multi-array immunoassay device such as time-cost, reproducibility and specificity, etc. were also investigated, respectively. The detection range for AFP, CEA, PSA and CA125 obtained by multi-array Pz immunosensor were 20-640 ng/ml, 1.5-30 microg/ml, 1.5-40 ng/ml and 5-150 IU/ml, respectively, with the coefficient of variance (CV) less than 5% and no cross-reactivates with other tumor markers in serum were observed. Application of the multi-array immunosensor to clinical samples demonstrated that results were in good agreement with chemiluminescence immunoassay (CLIA). Moreover, the multi-array Pz immunosensor could be regenerated to be reused for three cycles without appreciable loss of response activity. Therefore, the proposed multi-array immunoassay device based on Pz immunosensor provides a rapid, sensitive, specific, reusable, convenient and reliable alternative for the detection of tumor markers in clinical laboratory.     

Simultaneous determination of free mycophenolic acid and its glucuronide in serum of patients under mycophenolate mophetil therapy by ion-pair reversed-phase liquid chromatography with diode array UV detection

An high performance liquid chromatography (HPLC)-UV method for the simultaneous determination of the free forms of mycophenolic acid (MPA) and its phenol glucuronide (MPAG) in human serum samples was developed for the first time. Chromatographic separation was performed on octadecylsilane based stationary phase in combination with a mobile phase of methanol/buffered tetrabutylammonium (TBA) salt mixture. Sample pretreatment consisted of an ultrafiltration step followed by clean-up/enrichment on a C(18) solid-phase extraction (SPE) cartridge. Average recoveries of (99.7 +/- 0.2)% and (64.1 +/- 6.9)% for free MPA and MPAG, respectively, were estimated in the concentration range from 0.5 to 10 microg/ml. The within-day and between-days coefficients of variation were 0.4 and 0.8% for free MPA (0.1 microg/ml spiking level) and 0.8 and 1.6% for free MPAG (5 microg/ml spiking level), respectively. The linear ranges for free MPA and MPAG were 0.06-1 and 0.2-10microg/ml, respectively. Detection limits of 4 and 17 ng/ml for free MPA and MPAG were estimated in spiked serum. The same HPLC method was also capable of a simultaneous determination of the total concentration of MPA and MPAG when coupled to a proper sample pretreatment step. The potential of the method is demonstrated by excretion kinetics measurement in serum of patients receiving MMF therapy.     

Simultaneous LC-MS-MS determination of cyclosporine A, tacrolimus, and sirolimus in whole blood as well as mycophenolic acid in plasma using common pretreatment procedure

The purpose of the study was to develop rapid and simple procedure for simultaneous determination of cyclosporine A (CsA), tacrolimus (TCR), and sirolimus (SIR) in whole blood and mycophenolic acid (MPA) in plasma. Ascomycin (ASCO), cyclosporine D (CsD), and desmethoxysirolimus (DMSIR) were used as internal standards (IS) for TCR, CsA and MPA, and SIR, respectively. In the method development, six-level blood calibrators were used for CsA (range 47-1725 ng/ml), TCR (range 2.1-38.8 ng/ml), and SIR (range 2.4-39.6 ng/ml). Four-level calibrators were used for MPA (range 0.15-5.48 microg/ml). Four levels of quality control (QC) standards were used for blood samples, together with two levels of QC standards in plasma. All QC standards and calibrators were obtained from commercial sources. Sample preparation based on precipitation of 50 microl of sample in zinc sulfate-methanol-acetonitrile mixture containing IS, followed by centrifugation. HPLC was performed on ChromSpher pi column, 30 mm x 3 mm, in ballistic gradient of ammonium formate buffer-methanol at 0.8 ml flow rate. Following gradient elution profile was applied: 0-1.2 min at 30% methanol (divert valve to waste), 1.21-3.1 min 97% methanol (divert valve to detector), 3.11-3.7 min 30% methanol (divert valve to waste). ESI-MS-MS (MRM) was done on TSQ Quantum instrument with ESI source in positive ion mode. Ammoniated adducts of protonated molecules were used as precursor ions for all analytes but MPA. For this compound sodium adduct was used. Following transitions were monitored: for CsA m/z 1220-1203; for CsD 1234-1217; for SIR 931.6-864.5 and 882.6; for DMSIR 902-834.5; for TCR 821.5-768.5 and 785.5; for ASCO 809.5-756; for MPA 343-211.6; for MPA-glucuronide 514-306 and 211.6. The limits of quantitation were: 1 ng/ml for TCR and SIR, 20 ng/ml for CsA, and 0.1 microg/ml for MPA. Post-column infusion experiments showed that no positive or negative peaks appeared after injection of matrix in the elution range of target compounds. General signal suppression caused by matrix ranged from 20-40%, and was caused mainly by zinc sulfate present in deproteinizing solution. Extracted samples were stable for 2 days at 4 degrees C and for at least 20 days at -20 degrees C. MPA was fully separated from its glucuronide, which was eluted at around 0.7-0.8 min and directed to the waste. Some mutual cross-contribution of CsD and CsA was observed (below 1%), other IS did not contribute to target compounds and vice versa. Observations of chromatograms from patients taken single therapy demonstrated that possible metabolites of CsA, TCR, or SIR did not interfere with target compounds or IS.     

Type 2 diabetes mellitus in a non-obese mouse model induced by Meg1/Grb10 overexpression

We assessed the possibility of C57BL/6-Tg (Meg1/Grb10)isn(Meg1 Tg) mice as a non-obese type 2 diabetes (2DM) animal model. Meg1 Tg mice were born normal, but their weight did not increase as much as normal after weaning and showed about 85% of normal size at 20 weeks of age. Body mass index of Meg1 Tg mice was also smaller than that of control mice. The glucose tolerance test and insulin tolerance test showed that Meg1 Tg mice had reduced ability to normalize the blood glucose level. Blood urea nitrogen (BUN) in Meg1 Tg mice (19.6 +/- 1.2 mg/dl) was significantly lower than in controls (22.0 +/- 0.8 mg/dl), while plasma triglyceride, insulin, adiponectin, and resistin levels were significantly higher (202.0 +/- 23.4 mg/dl vs 146.3 +/- 23.4 mg/dl, 152.4 +/- 16.3 pg/ml vs 88.1 +/- 16.9 pg/ml, 74.4 +/- 10.9 microg/ml vs 48.3 +/- 7.0 microg/ml, and 4.0 +/- 0.2 ng/ml vs 3.6 +/- 0.2 ng/ml, respectively). Body, visceral fat weight and liver weights were significantly lower (19.6 +/- 0.4 g vs 24.3 +/- 0.3 g, 376.7 +/- 29.6 mg to 507.5 +/- 23.0 mg, and 906.0 +/- 41.8 mg to 1,001.0 +/- 15.1 mg, respectively). Thus, hyperinsulinemia observed in Meg1 Tg mice indicates that their insulin signaling pathway is somehow inhibited. With high fat diet, the diabetes onset rate of Meg1 Tg mice increased up to 60%. These results suggest that Meg1 Tg mice resemble human 2DM.     

Serum follicle stimulating hormone, luteinizing hormone, and testosterone in sexually stimulated intact and unilaterally castrated rams

Ten two-year-old intact (IN) and unilaterally castrated (UC) Targhee rams were exposed to an estrogenized ewe each week from June to October. Each week the rams were subjectively evaluated for libido (10 for high interest and 1 for no interest). Semen was collected from all cooperating rams and evaluated for volume, concentration, and motility. Every 2 wk, blood samples were obtained at -30 and 0 min before and 30 and 60 min after ewe access. Serum was harvested; follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone concentrations were quantified by radioimmunoassay (RIA). Week 5 of ewe access was assigned as Week 1. Libido scores rose from a low on Week 1, with eight rams ejaculating, to a high on Week 12, with all rams ejaculating (Week 1, 5.0 +/- 1.0; Week 12, 10.0 +/- 0.0). The product of testis length and width was significantly greater in UC compared with IN rams (88.4 +/- 1.4 versus 73.2 +/- 1.0 cm(2), respectively). Serum FSH concentrations (ng/ml) were greater (P < 0.05) in UC than IN rams and dropped over the experimental period. Serum LH concentrations (ng/ml) were significantly greater in UC compared with IN rams. This difference was more pronounced in Weeks 1 and 3 compared with Weeks 11 and 13. Serum testosterone concentrations (ng/ml) were similar in UC and IN rams throughout the experiment. In conclusion, serum testosterone was not altered in UC rams; however, serum FSH and LH concentrations were increased in UC rams. Unilateral castration did not enhance the normal changes in semen quantity and quality in the rams from July to October.     

Improved RP-HPLC method to determine neferine in dog plasma and its application to pharmacokinetics

Existing methods to determine neferine, a bisbenzylisoquinline alkaloid, either have no internal standard or lack selectivity, or take longer time. Here an improved reverse-phase high-performance liquid chromatographic (RP-HPLC) method was established in biological samples. The extraction recovery was 90.9% for neferine at concentration level of 0.2 microg/ml and 77.7% for dauricine (the internal standard) at 5 microg/ml in dog plasma, respectively. The linear quantification range of the method was 25-2000 ng/ml in dog plasma, with linear correlation coefficients greater than 0.999. The intra-day and inter-day relative standard deviations (R.S.D.s) for neferine at 50, 200 and 1000 ng/ml levels in dog plasma fell in the range of 3.0-5.4% and 4.3-9.5%, respectively. The RP-HPLC method was successfully applied to a pharmacokinetics study, in which experimental dogs received a single dose of neferine (5 mg/kg i.v. or 10 mg/kg p.o.). The pharmacokinetic result was presented.     

Determination of disodium clodronate in human plasma and urine using gas-chromatography-nitrogen-phosphorous detections: validation and application in pharmacokinetic study

We present a specific method for the determination of disodium clodronate in human plasma and urine using a gas-chromatographic system with nitrogen phosphorus detector (NPD). The compound was extracted from plasma and urine samples by an anion-exchange resin and derivatizated with bistrimethylsilyltrifluoroacetamide (BSTFA). Sodium bromobisphosphonate was used as internal standard. The calibration curves were linear in both plasma and urine, with a regression coefficient r > 0.9975 in plasma and r > 0.9977 in urine.The limit of quantitation was 0.3 microg/ml in plasma and 0.5 microg/ml in urine. The method was validated by intra-day assays at three concentration levels. During the study we carried out inter-day assays to confirm the feasibility of the method. The precision in plasma at 0.5, 15, and 45 microg/ml was 12.4, 0.2, and 6.5% (n = 40), respectively; in urine at 0.8, 8, and 40 microg/ml it was 8.6, 6.4, and 9.3% (n = 40), respectively.The method was accurate and reproducible, and was successfully applied to determine the pharmacokinetic parameters of clodronate in healthy volunteers after intravenous infusion and intramuscular injection of 200 mg of the compound. The Cmax after intravenous infusion and intramuscular injection was 16.1 and 12.8 microg/ml, respectively. AUC(0-48 h) after infusion administration and intramuscular injection was 44.2 +/- 18.0 and 47.5 +/- 12.4 h microg/ml, respectively. The elimination half-life in both administrations was 6.31 +/- 2.7 h.     

Changes in the tumor marker concentration in female patients with hyper-, eu-, and hypothyroidism

The levels of 6 circulating tumor markers were evaluated in a total of 131 female subjects with altered thyroid states; 36 normal subjects, 46 hyperthyroid patients with Graves' disease, and 49 primary hypothyroid patients. The mean CEA concentration was observed to be significantly higher (p less than 0.02) in hypothyroid patients than in normal and hyperthyroid patients (1.1 +/- 0.1 ng/ml, 0.8 +/- 0.1 ng/ml and 0.8 +/- 0.1 ng/ml, respectively). Similarly, the mean serum CA 125 concentration in hypothyroid patients was higher (p less than 0.02) than in normal and hyperthyroid patients (13.0 +/- 2.6 U/ml, 7.6 +/- 1.1 U/ml and 5.5 +/- 0.8 U/ml, respectively), and the mean serum CA 15-3 concentration in hypothyroid patients was significantly higher than in normal subjects (p less than 0.01) and hyperthyroid patients (p less than 0.001) (16.2 +/- 0.9 U/ml, 13.9 +/- 0.6 U/ml and 10.6 +/- 0.5 U/ml, respectively). No statistical difference was found in mean CA 19-9 in the three subject groups. AFP in the hypothyroid patients (3.6 +/- 0.3 ng/ml) was significantly higher (p less than 0.05) than in normal subjects (2.6 +/- 0.2 ng/ml) and hyperthyroid patients (1.7 +/- 0.2 ng/ml) (p less than 0.01). On the other hand, serum ferritin was low in the hypothyroid patients (65.9 8.0 ng/ml) and significantly increased (69.1 +/- 9.0 ng/ml) (p less than 0.02) with the normalization of thyroid function. In hyperthyroidism, serum ferritin (70.2 +/- 7.0 ng/ml) was significantly higher than in the hypothyroid patients (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of betaxolol in human aqueous humour by high-performance liquid chromatography with fluorescence detection

A reversed-phase high-performance liquid chromatographic method is described for the determination of betaxolol in human aqueous humour. Betaxolol and the internal standard metoprolol were extracted with cyclohexane and separated on a reversed-phase column (Luna C(18), 250 x 4.6 mm, 5 microm) with a mobile phase containing acetonitrile-phosphate buffer (40:60, v/v) at a flow-rate of 0.8 ml/min. The column effluent was monitored with a fluorescence detector at 227 nm (excitation) and 301 nm (emission). The retention times for metoprolol and betaxolol were 3.55 and 5.63 min, respectively. The recovery from aqueous humour was found to be 71.6% for betaxolol at 1.25 microg/ml. The within-day and day-to-day accuracy values were in the range of 96.17-105.2% for betaxolol at 0.1, 4 and 12 microg/ml (n=6), within-day and day-to-day precision values were less than 10% for betaxolol at the concentrations given above. The detection limit corresponding to the signal-to-noise ratio of 3:1 was 15 ng/ml. The presented method was suitable for measuring betaxolol levels in human aqueous humour samples obtained from patients after topical administration.     

High-performance liquid chromatographic analysis of bisphenol A and 4-nonylphenol in serum, liver and testis tissues after oral administration to rats and its application to toxicokinetic study

A sensitive and simple method based on solid-phase extraction (SPE) and HPLC with fluorescence detection for the determination of bisphenol A (BPA) and 4-nonylphenol (4-NP) in rat serum, liver and testis tissues has been developed. The chromatographic conditions consisted of a C18 column and mobile phase composition of acetonitrile and water with flow rate of 1.0 ml/min. The fluorescence detection was performed at excitation and emission wavelengths of 227 nm and 313 nm, respectively. Under these conditions, BPA and 4-NP were well separated and showed good linearities in the ranges of 0.01-50.0 microg/ml for BPA and 0.15-150.0 microg/ml for 4-NP with correlation coefficients greater than 0.999. The detection limits of serum and tissue samples were 2.8 ng/ml and 1.4 ng/g for BPA and 5.6 ng/ml and 2.8 ng/g for 4-NP at a signal-to-noise ratio (S/N) of 3. The intra-assay and the inter-assay precisions were better than 11.4%. Recoveries of BPA and 4-NP were 78.6-95.0% and 80.2-93.4%, respectively. The proposed method was applied to a toxicokinetic study of BPA and 4-NP including individual and combined oral administration to rats. The results showed that 4-NP remarkably altered the toxicokinetic parameters of BPA in testis, while parameters of BPA were not obviously altered in serum and liver under the experimental conditions investigated. On the other hand, there was no significant difference in the toxicokinetics of 4-NP when administered with BPA.     

Effects of growth hormone and insulin-like growth factor-I on development of in vitro derived bovine embryos

The objectives of this study were to determine whether the addition of growth hormone (GH) to maturation medium and GH or insulin-like growth factor-I (IGF-I) to culture medium affects development of cultured bovine embryos. We matured groups of 10 cumulus-oocyte complexes (COCs) in serum-free TCM-199 medium containing FSH and estradiol with or without 100 ng/ml GH. After fertilization, we transferred groups of 10 putative zygotes to 25 microl drops of a modified KSOM medium containing the following treatments: non-specific IgG (a control antibody, 10 microg/ml); GH (100 ng/ml) + IgG (10 microg/ml, GH/IgG); IGF-I (100 ng/ml) + IgG (10 microg/ml, IGF/IgG); antibody to IGF-I (10 microg/ml, anti-IGF); GH (100 ng/ml) + anti-IGF (10 microg/ml GH/anti-IGF); IGF-I (100 ng/ml) + anti-IGF (10 microg/ml, IGF/anti-IGF); no further additions (control). We repeated the experiment six times. Adding GH to the maturation medium increased cleavage rates at Day 3 compared to control (87.3 +/- 1.2% > 83.9 +/- 1.2%; P < 0.05) but had no effects on blastocyst development at Day 8. At Day 8, blastocyst development was greater (P < 0.01) for GH/IgG (24.8 +/- 2.5%) and IGF/IgG (33.7 +/- 2.5%) than for IgG (16.1 +/- 2.1%) and greater for IGF/IgG than for GH/IgG (P < 0.02). Blastocyst development at Day 8 did not differ between anti-IGF (20.4 +/- 1.8%) and GH/anti-IGF (24.1 +/- 1.9%) or IGF/anti-IGF (17.7 +/- 1.9%), but it was greater for GH/anti-IGF than for IGF/anti-IGF (P < 0.05). The Day 8 blastocysts of GH/IgG and IGF-I/IgG groups had a higher (P < 0.01) number of cells than the IgG group. The addition of anti-IGF-I eliminated the effects of IGF-I on cell number but did not alter GH effects. In conclusion, both GH and IGF-I stimulate embryonic development in cattle and GH effects may likely involve IGF-I-independent mechanisms.     

Simultaneous determination and pharmacokinetic studies of dihydromyricetin and myricetin in rat plasma by HPLC-DAD after oral administration of Ampelopsis grossedentata decoction

A simple and sensitive high-performance liquid chromatographic method was developed for the simultaneous determination of dihydromyricetin (1) and myricetin (2) in rat plasma after orally administrating the decoction of Ampelopsis grossedentata. Plasma samples were acidified with 0.375% phosphoric acid and extracted with ethyl acetate. Analysis of the extract was performed on reversed-phase C(18) column with a gradient eluent composed of acetonitrile and 0.04% phosphoric acid. The flow rate was kept at 1 ml/min and the detection wavelength was set at 290 and 370 nm for 1 and 2, respectively. The calibration curves were linear in the range of 0.247-4.114 microg/ml and 0.150-2.501 microg/ml for 1 and 2, respectively. The intra-day and inter-day precisions were better than 4.9 and 6.2%, respectively. The limits of detection (LOD) for 1 and 2 in plasma were 21.600 and 52.530 ng/ml, and the limits of quantification (LOQ) were 0.247 and 0.150 microg/ml, respectively. The mean recoveries for 1 and 2 were 92.0 and 93.3%, respectively. The accuracy and precision were well within the acceptable range and R.S.D. of measured rat samples was less than 7.5%. This validated method has been successfully applied in the pharmacokinetics study of dihydromyricetin and myricetin in vivo after orally administrating the decoction of A. grossedentata to rats.     

Determination of amiodarone and desethylamiodarone in horse plasma and urine by high-performance liquid chromatography combined with UV detection and electrospray ionization mass spectrometry

A rapid method for the quantification of amiodarone and desethylamiodarone in animal plasma using high-performance liquid chromatography combined with UV detection (HPLC-UV) is presented. The sample preparation includes a simple deproteinisation step with acetonitrile. In addition, a sensitive method for the quantification of amiodarone and desethylamiodarone in horse plasma and urine using high-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is described. The sample preparation includes a solid-phase extraction (SPE) with a SCX column. Tamoxifen is used as an internal standard for both chromatographic methods. Chromatographic separation is achieved on an ODS Hypersil column using isocratic elution with 0.01% diethylamine and acetonitrile as mobile phase for the HPLC-UV method and with 0.1% formic acid and acetonitrile as mobile phase for the LC-MS/MS method. For the HPLC-UV method, good linearity was observed in the range 0-5 microg ml(-1), and in the range 0-1 microg ml(-1) for the LC-MS/MS method. The limit of quantification (LOQ) was set at 50 and 5 ng ml(-1) for the HPLC-UV method and the LC-MS/MS method, respectively. For the UV method, the limit of detection (LOD) was 15 and 10 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs of the LC-MS/MS method in plasma were much lower, i.e. 0.10 and 0.04 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs obtained for the urine samples were 0.16 and 0.09 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The methods were shown to be of use in horses. The rapid HPLC-UV method was used for therapeutic drug monitoring after amiodarone treatment, while the LC-MS/MS method showed its applicability for single dose pharmacokinetic studies.     

Inhibin production by macaque granulosa cells from pre- and periovulatory follicles: regulation by gonadotropins and prostaglandin E2.

Although inhibin (IN) is secreted by granulosa cells (GC) of preovulatory follicles, the major source of immunoreactive IN circulating during the primate ovarian cycle is the corpus luteum. The aims of this study were (1) to investigate culture conditions for optimal IN production by luteinized GC (LGC) from rhesus monkeys and (2) to compare IN and progesterone (P) production by nonluteinized GC (NGC) and LGC in response to putative agonists. Animals were treated for up to 9 days with human menopausal gonadotropins to promote the development of multiple preovulatory follicles. GC were obtained from large follicles before (NGC) or 27 h after (LGC) an ovulatory injection of hCG. For Aim 1, cells were cultured in Hams F-10 medium +/- hCG (100 ng/ml) with or without the addition of insulin/transferrin/selenium, 10% fetal bovine serum, or 10% Serum-Plus (JRH Biosciences, Lenexa, KS). Medium was changed on Days 1, 2, 4, 6, and 8, and IN and P concentrations were determined by RIA. Basal (unstimulated) IN production by LGC was enhanced and maintained for 6-8 days in the presence of serum, but rapidly declined in the absence of serum. In contrast, basal P secretion declined regardless of exposure to serum. Human CG consistently increased (p less than 0.05) IN production only in the presence of serum but stimulated (p less than 0.05) P production under all conditions. For Aim 2, cells were cultured for 4 days in Ham's F-10 medium + 10% macaque serum +/- hCG (100 ng/ml), hFSH (100 ng/ml), prostaglandin E2(PGE2; 14 microns), or dibutyryl(db)-cAMP (5 mM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of corticosterone and 17-hydroxycorticosterone in plasma and urine samples by sweeping techniques using micellar electrokinetic chromatography

The analysis of corticosterone in mouse blood serum (metabolic-stress experiment) and 17-hydroxycorticosterone in human urine (exercise-stress experiment) samples by means of capillary electrophoresis/UV absorbance in conjunction with online sample concentration techniques is described. The use of normal MEKC had an analyte detection limit of 7 microg/ml (S/N=3); whereas when online sample concentration methods, including sweeping-micellar electrokinetic chromatography (Sweeping-MEKC) and cation-selective exhaustive injection-sweep-micellar electrokinetic chromatography (CSEI-sweep-MEKC) were used, the detection limits could be improved to 3 and 5 ng/ml, respectively. In the analysis of actual samples from animal metabolic-stress experiments (39 mouse), chronically stressed animals showed a higher level (552+/-152 ng/ml) and acute stressed animals showed an intermediate level (375+/-105 ng/ml). In comparison, normal animals show a lower concentration level of corticosterone (153+/-109 ng/ml). In addition, based on a human exercise-stress experiment (seven volunteers), the acute stressed humans (after exercise, 800 m of running) show a higher concentration of 17-hydroxycorticosterone (113+/-55 ng/ml for males; 128+/-25 for females) and the non-stressed humans (before exercise) show a lower concentration (63+/-37 ng/ml for male; 60+/-20 for female), respectively.     

Determination of paraldehyde by gas chromatography in whole blood from children

A rapid, sensitive and selective gas chromatographic method with flame ionization detection was developed for the determination of paraldehyde in small blood samples taken from children. Whole blood samples (300 microl) collected in a 3 ml Wheaton glass sample vial were spiked with acetone (internal standard: 15 ng) followed by addition of concentrated hydrochloric acid. The mixture was heated in the sealed airtight sample vial in a water bath (96 Celsius; 5 min) to depolymerize paraldehyde to acetaldehyde. A 2 ml aliquot of the headspace was analyzed by gas chromatography with flame ionization detector using a stainless steel column (3 m x 4 mm i.d.) packed with 10% Carbowax 20 M/ 2% KOH on 80/100 Chromosorb WAW. Calibration curves were linear from 1.0-20 microg (r2>0.99). The limit of detection was 1.5 microg/ml, while relative mean recoveries at 2 and 18 microg were 105.6 +/- 8.4 and 101.2 +/- 5.9%, respectively (n = 10 for each level). Intra- and inter-assay relative standard deviations at 2, 10 and 18 microg were <15%. There was no interference from other drugs concurrently used in children with severe malaria, such as anticonvulsants (diazepam, phenytoin, phenobarbitone), antipyretics/analgesics (paracetamol and salicylate), antibiotics (gentamicin, chloramphenicol, benzyl penicillin) and antimalarials (chloroquine, quinine, proguanil, cycloguanil, pyrimethamine and sulfadoxine). The method was successfully applied for pharmacokinetic studies of paraldehyde in children with convulsions associated with severe malaria.     

Detection and quantification of aripiprazole and its metabolite, dehydroaripiprazole, by gas chromatography-mass spectrometry in blood samples of psychiatric patients

Aripiprazole is a novel antipsychotic drug for the treatment of schizophrenia and schizoaffective disorders. In this study, a new method using gas chromatography-mass spectrometry (GC-MS) was developed and validated for the detection of aripiprazole and its main metabolite, dehydroaripiprazole, in plasma. Blood samples from seven psychiatric patients treated with aripiprazole (10-20 mg/day) underwent a solid-phase extraction (SPE) and N-methyl-N-trimethylsilytrifluoroacetamide (MSTFA) derivatization. The characteristic ions of mass spectra for aripiprazole and dehydroaripiprazole were m/z 306, 292, 218 and 304, 290, 218, respectively. Extraction recoveries from this method were 75.4% (n=5) for aripiprazole and 102.3% (n=5) for dehydroaripiprazole. The calibration curves of aripiprazole and dehydroaripiprazole were linear from 16 to 500 ng/ml (r(2)=0.999) and 8 to 250 ng/ml (r(2)=0.999), respectively. The respective limits of quantification (LOQs) for aripiprazole and dehydroaripiprazole evaluated in 0.5 ml of serum were 14.4 ng/ml and 6.9 ng/ml. Intra-assay and interassay precision and accuracy were within acceptable ranges. In this study, we also found that the mean trough concentrations in plasma at steady-state were 128.9 microg/l for aripiprazole and 30.1 microg/l for dehydroaripiprazole.     

Pharmacokinetic study of ellagic acid in rat after oral administration of pomegranate leaf extract

Quantification of ellagic acid, the principal bioactive component of pomegranate leaf extract, in rats plasma following oral administration of pomegranate leaf extract was achieved by using a high-performance liquid chromatographic method. The calibration curve for ellagic acid was linear (r2=0.9998) ver the concentration range 0.026-1.3 microg/ml. The intra- and inter-day assays of ellagic acid from rat plasma were less than 6.52% at concentration range from 26 to 1300 ng/ml and good overall recoveries (94.5-102.4%) were found on same concentrations. The concentration-time profile was fitted with an open two-compartment system with lag time and its max concentration of ellagic acid in plasma was 213 ng/ml only 0.55 h after oral administration extract 0.8 g/kg. The pharmacokinetic profile indicates that ellagic acid has poor absorption and rapid elimination after oral administration pomegranate leaf extract, and part of it was absorbed from stomach.     

Plasma transport and tissue distribution of beta-carotene, vitamin A and retinol-binding protein in domestic cats.

The objective of this study was to determine retinol, retinyl esters and retinol-binding protein (RBP) as well as carotenoids in plasma, urine, liver and kidneys of randomly selected domestic cats. Retinol (240+/-64 ng/ml, mean+/-S.D.) represented one-third of total retinyl esters (736+/-460 ng/ml) in plasma. Retinyl esters were stearate, palmitate and oleate representing 61+/-6, 36+/-13 and 5+/-3% of total retinyl esters, respectively. In half of the cats, retinyl esters (22+/-21 ng/ml) were found in the urine. Vitamin A in the livers (4317+/-1956 microg/g) was significantly higher than in the kidney cortex and medulla (14.16+/-8.92 and 7.59+/-4.52 microg/g, respectively, both P<0.001). RBP was detected in the plasma but not in the urine. Immunoreactive RBP was observed in hepatocytes and in the cells of the proximal tubules. beta-Carotene was present in plasma but never in tissues. The results show that similar to canines differences in vitamin A metabolism in cats are related to the occurrence of retinyl esters in plasma. They differ, however, with regard to the tissue distribution of beta-carotene and the excretion of vitamin A in the urine.