Gas chromatographic negative ion mass spectrometric assay of 2-dicyclopropylmethylamino-2-oxazoline (S-3341), a new antihypertensive drug

The quantification in plasma and urine of 2-dicyclopropylmethylamino-2-oxazoline (S-3341), a new antihypertensive drug is described using a sensitive gas chromatographic negative ion mass spectrometric method with ammonia as moderating gas. After a two-step extraction, derivatization is carried out with 3,5-bis(trifluoromethyl)benzoyl chloride and the abundance of the molecular ion (m/z 420) obtained is compared with that of the tetradeuterated standard (m/z 424). The low background due to the high mass and negative ion detection provides a detection limit of about 1 pg per injection. Oral administration of 1 or 2 mg S-3341 to patients gives a maximum concentration of 3.3 +/- 0.7 ng ml-1 and 7.6 +/- 2.0 ng ml-1 at 1.8 +/- 0.6 h and 1.4 +/- 0.7 h and an average elimination half-life of 6.7 h.     

Determination of tegaserod by LC-ESI-MS/MS and its application to a pharmacokinetic study in healthy Chinese volunteers

A simple, rapid and sensitive high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) assay for determination of tegaserod in human plasma using diazepam as internal standard (IS) was established. After adjustment to a basic pH with sodium hydroxide, plasma was extracted by ethyl acetate and separated by high performance liquid chromatography (HPLC) on a reversed-phase C18 column with a mobile phase of methanol: 5 mM ammonium acetate (75:25, v/v, adjusting the pH to 3.5 with glacial acetic acid). The quantification of target compounds was obtained by using multiple reaction monitoring (MRM) transitions; m/z 302.5, 173.2 and 285.4, 193.2 were measured in positive mode for tegaserod and internal standard (diazepam), respectively. The lower limit of quantification (LLOQ) was 0.05 ng/ml. The calibration curves were linear over the range 0.05-8.0 ng/ml (r=0.9996) for tegaserod. The mean absolute recovery of tegaserod was more than 85.56%. Intra- and inter-day variability values were less than 9.21% and 10.02%, respectively. The samples were stable for 8h under room temperature (25 degrees C, three freeze-thaw cycles in 30 days and for 30 days under -70 degrees C). After administration of a single dose of tegaserod maleate 4 mg, 6 mg and 12 mg, respectively, the area under the plasma concentration versus time curve from time 0 h to 12 h (AUC0-12) were (2.89+/-0.88), (5.32+/-1.21) and (9.38+/-3.42) ng h/ml, respectively; peak plasma concentration (Cmax) were (1.25+/-0.53), (2.21+/-0.52) and (4.34+/-1.66) ng/ml, respectively; apparent volume of distribution (Vd/F) were (6630.5+/-2057.8), (7615.2+/-2242.8) and (7163.7+/-2057.2) l, respectively; clearance rate (CL/F) were (1851.4+/-496.9), (1596.2+/-378.5) and (1894.2+/-459.3) l/h, respectively; time to Cmax (Tmax) were (1.00+/-0.21), (1.05+/-0.28) and (1.04+/-0.16) h, respectively; and elimination half-life (t1/2) were (3.11+/-0.78), (3.93+/-0.92) and (3.47+/-0.53) h, respectively; MRT were (3.74+/-0.85), (4.04+/-0.56) and (3.28+/-0.66) h, respectively. The essential pharmacokinetic parameters after oral multiple doses (6mg, b.i.d) were as follows: Cssmax, (2.72+/-0.61) ng/ml; Tmax, (1.10+/-0.25) h; Cssmin, (0.085+/-0.01) ng/ml; Cav, (0.54+/-0.12) ng/ml; DF, (4.84+/-0.86); AUCss, (6.53+/-1.5) ngh/ml. This developed and validated assay method had been successfully applied to a pharmacokinetic study after oral administration of tegaserod maleate in healthy Chinese volunteers at a single dose of 4 mg, 6 mg and 12 mg, respectively. The pharmacokinetic parameters can provide some information for clinical medication.     

HPLC-APCI-MS for the determination of vitamin K(1) in human plasma: method and clinical application

A sensitive HPLC-APCI-MS method for the determination of vitamin K(1) (VK-1) in human plasma was established. Target ions at [M+H](+)m/z 451.5 for VK-1 and [M+H](+)m/z 331.4 for the I.S. (teprenone). Calibration curve was linear over the range of 0.3-1,000 ng/ml. The lower limit of quantification was 0.3 ng/ml. The intra- and inter-batch variability values were less than 8% and 15%, respectively. The C(max) was 210.1+/-86.7 ng/ml while the elimination half-life (t(1/2)) was 8.8+/-1.7h and time to the C(max) was 5.5+/-0.8h after administration of soft capsule containing 10mg VK-1.     

Simultaneous determination of glipizide and rosiglitazone unbound drug concentrations in plasma by equilibrium dialysis and liquid chromatography-tandem mass spectrometry

Glipizide and rosiglitazone are widely used to treat Type 2 diabetes. In order to investigate drug-drug protein binding interaction between glipizide and rosiglitazone, a method was developed and validated for simultaneously determining the free (unbound) fraction of glipizide and rosiglitazone in plasma employing equilibrium dialysis for the separation of free drug and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for quantitation. Post-dialysis human plasma or buffer samples of 0.2 ml were extracted using a liquid-liquid extraction procedure and analyzed by a high performance liquid chromatography electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a Zorbax SB-Phenyl column, ionized using an atmospheric pressure electrospray ionization source and analyzed in positive ion mode with multiple reaction monitoring. The ion transitions monitored were m/z 446-->321 for glipizide, m/z 358-->135 for rosiglitazone, and m/z 271-->155 for tolbutamide (internal standard, IS). The chromatographic run time was 5 min per injection, with retention times of 2.3, 3.4 and 2.3 min for glipizide, rosiglitazone and IS, respectively. The calibration curves of glipizide and rosiglitazone were over the range of 1-2000 ng/ml (r(2)>0.9969) in the combined matrix of human plasma and isotonic sodium phosphate buffer (1:1, v/v). The inter-assay precision and accuracy of the quality control samples were <10.9% of coefficient of variability and >93.5% and 94.5% of nominal concentration for glipizide and rosiglitazone, respectively. The lower limit of quantitation of both glipizide and rosiglitazone was 1.0 ng/ml. Both glipizide and rosiglitazone bound to plasma protein extensively (>99% bound). Glipizide and rosiglitazone free fraction averaged 0.678+/-0.071 and 0.389+/-0.061%, respectively, at plasma concentration of 1000 ng/ml. This developed method proves reproducible and sensitive and its application to clinical samples is also reported.     

Glucocorticoid effects on the diurnal rhythm of circulating leptin levels

It is known that circulating leptin shows diurnal variation with a nocturnal rise; however, the mechanisms generating this rhythm have not been fully elucidated. Glucocorticoids are a potent stimulator of leptin secretion, and there is a reciprocal relationship between circulating leptin and glucocorticoid levels. We hypothesized that glucocorticoids could modulate the diurnal rhythm of circulating leptin. We therefore explored the diurnal variation of leptin under situations in which subjects showed no or some shift of glucocorticoid diurnal rhythm, such as prednisolone-administered humans, and adrenalectomized and corticosterone-replaced (ADX+B) rats. The peak level of plasma cortisol immunoreactivity was shifted from early morning to noon by prednisolone administration. The nocturnal increment of plasma leptin in prednisolone-administered patients (71.2 +/- 14.2% from 08:00 h value) was significantly greater than that in normal volunteers (12.2 +/- 7.5% from 08:00 h value), but the timing of nadir and the peak of plasma leptin was not shifted. In normal rats, the plasma concentration of leptin showed the diurnal rhythm with the bottom at 16:00 h and the top between midnight and early morning. The amplitude of leptin diurnal rhythm was significantly reduced in ADX+B rats (08:00 h: 3.0 +/- 0.2, 16:00 h: 2.7 +/- 0.2, 00:00 h; 3.7 +/- 0.2 ng/ml) compared with sham operated rats (08:00 h: 3.0 +/- 0.2, 16:00 h 2.2 +/- 0.2, 00:00 h: 4.7 +/- 0.4 ng/ml); but ADX+B rats still retained similar timing of nadir and the peak of plasma leptin as observed in sham rats. These results indicate that glucocorticoids enhance the amplitude of leptin diurnal rhythm, and are consistent with previous findings showing that glucocorticoids increase leptin secretion. Glucocorticoids appear to play modulatory, but not essential roles in generating leptin diurnal rhythm.     

Pituitary response of prepuberal lambs to oestradiol-17 beta.

In two experiments 48 prepuberal Merino ewe lambs were injected with oestradiol-17 beta (E2) or saline to study the effect of E2 on their plasma LH levels and on oestrus and ovulation. In the three groups which received 30 (experiment I), 50 and 30 (experiment II) microgram E2 respectively, 27 out of 28 lambs showed an LH response, the corresponding mean LH peaks being 64.3 +/0 22.5, 153.6 +/-33.4 and 91.7 +/- 16.9 ng/ml at mean intervals of 11.1, 11.2 and 10.5 h, respectively, after injection. None of the 20 lambs in the control groups had an LH level higher than 18 ng/ml 12 h after injection. In the three E2 groups, 41.7, 62.5 and 37.5% of animals showed oestrus within 26 h of injection while in the control groups only one animal showed oestrus. Of 13 animals showing oestrus in the E2 groups, 11 failed to ovulate. The mean pre-injection plasma FSH level in experiment I was 102.7 ng/ml, and in four 5--7-month-old lambs over several weeks uas 155.3 ng/ml. Despite these high pre-injection levels of FSH, it appears that the follicles were unable to respond to the LH peak which followed the E2 injection.     

The determination of gemcitabine and 2'-deoxycytidine in human plasma and tissue by APCI tandem mass spectrometry

A fast, sensitive and accurate method for the determination of gemcitabine (difluorodeoxycytidine; dFdC) and deoxycytidine (CdR) in human plasma/tissue was developed using LC-MS/MS techniques. Effectiveness of the method is illustrated with the analysis of plasma from a phase I trial of dFdC administered as a 24h infusion. The method was developed using (15)N(3) CdR as an internal standard across the concentration range of 1-500ng/ml, using a cold alcohol-protein precipitation followed by desorption with freeze drying. Sample clean-up for LC-MS/MS analysis was performed by an innovative liquid/liquid back extraction with ethyl acetate and water. Chromatography was performed using a Chrompak-spherisorb-phenyl-column (3.1mmx200mm, 5microm) with a 50mM formic acid: acetonitrile (9:1) mobile phase eluted at 1ml/min. Extracted samples were observed to be stable for a minimum of 48h after extraction when kept at 4 degrees C. Detection was performed using an atmospheric pressure chemical ionization (APCI) source and mass spectrometric positive multi-reaction-monitoring-mode (+MRM) for dFdC (264 m/z; 112 m/z), CdR (228 m/z; 112 m/z), and (15)N(3) CdR (231 m/z; 115 m/z) at an ion voltage of +3500V. The accuracy, precision and limit-of-quantitation (LOQ) were as follows: dFdC: 99.8%, +/-7.9%, 19nM; CdR: 100.0%, +/-5.3%, 22nM, linear range LOQ to 2microM. During 24h infusion dFdC levels were detected with no interference from either CdR or difluorodeoxyuridine (dFdU). CdR co-eluted with dFdC but selectivity demonstrated no "crosstalk" between the compounds. In conclusion the analytical assay was very sensitive, reliable and robust for the determination of plasma and tissue concentrations of dFdC and CdR.     

Effects of growth hormone-releasing factor and somatostatin on growth hormone secretion in hypophysial stalk-transected beef calves

The effects of growth hormone-releasing factor (GHRF) on growth hormone (GH) secretion were studied in beef calves after hypophysial stalk transection (HST). Peripheral GH concentration during surgery was elevated for 60 min after the initiation of anesthesia to 15 ng/ml, which was greater than plasma levels after HST and during the recovery period (0-30 hr mean, 3 ng/ml; P less than 0.05). Episodic GH secretion normally seen in sham-operated controls (SOC) was abolished after HST. Before HST, calves responded to 80% of the GHRF challenges, whereas after HST calves responded to every challenge of GHRF with an increase in plasma GH. A dose of 0.067 microgram human pancreatic (hp) hpGHRF(1-40)OH/kg body wt 3 days after HST increased plasma GH to 55 ng/ml from a control period mean of 5 ng/ml (P less than 0.04). On Day 8, HST calves received two injections of 0.067 microgram hpGHRF/kg body wt at 3-hr intervals, with feeding 70 min after the first injection. During two preinjection control periods, basal GH averaged less than 4 ng/ml and increased to 17 (P less than 0.02) and 9 (P less than 0.04) ng/ml immediately after the first and second injection of hpGHRF, but the response declined over the 8-day period after surgery. On Days 19 and 20, the HST calves were infused iv with 0.033 and 0.067 microgram somatostatin(SS)-14 (SRIH)/kg body wt, during which a pulse injection of 0.067 microgram hpGHRF/kg body wt was administered. GH increased to 9 and 5 ng/ml during the 0.033- and 0.067-microgram SRIH infusions after GHRF; no somatotropic rebound was observed after the SRIH was discontinued as was seen in the animals while the hypothalamic-hypophysial connections were intact. Five and six months after HST the responses to two analogs of rat hypothalamic GHRF were similar to those in SOC calves. These results indicate that HST calves responded to exogenous GHRF with an abrupt increase in plasma GH, but GH response to GHRF during SRIH infusion was greatly inhibited.     

Opioid action on luteinizing hormone secretion in newborn pigs: paradoxical effect of naloxone

German Landrace piglets, 6-7 days of age, received either saline (9 males, 8 females), 0.5 mg naloxone/kg body weight (7 males, 7 females), 2.0 mg naloxone/kg (7 males, 8 females) or 0.5 mg DADLE (potent leu-enkephalin analog)/kg (7 males, 7 females) through a catheter inserted into the jugular vein 2-4 days previously. Male or female piglets were allocated randomly, within litter, to the different experimental groups. Blood samples were withdrawn for a period of 240 min at 10-min intervals for the first 60 min following injection and at 20-min intervals for the rest of the test period. Piglets were separated from their mother via a detachable wall and were allowed to suckle every 50 min. DADLE failed to alter plasma levels of LH in both males and females. Naloxone induced a significant (P less than 0.01) decrease in LH concentrations in females 10 to 60 min after injection (saline: 2.3 +/- 0.2 ng/ml plasma (SEM); 0.5 mg naloxone/kg: 1.0 +/- 0.2 ng/ml plasma and 2 mg naloxone/kg 1.2 +/- 0.4 ng/ml plasma). In males low doses of naloxone reduced plasma LH levels 10 to 40 min after injection (saline: 2.0 +/- 0.3 ng/ml plasma and 0.5 ng naloxone/kg: 1.1 +/- 0.3 ng/ml), whereas a decrease in plasma LH levels occurred 80 to 140 min after injection of high doses of naloxone (saline: 2.1 +/- 0.2 ng/ml and 2 mg naloxone/kg: 1.0 +/- 0.2 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression of plasma FSH concentrations with bovine follicular fluid blocks ovulation in GnRH-treated seasonally anoestrous ewes

The specific requirement for FSH in the final stages of preovulatory follicle development was assessed in seasonally anoestrous ewes given 2-h injections of GnRH (250 ng/injection), with (N = 10) or without (N = 10) concurrent treatment with bovine follicular fluid (bFF: 2 ml given i.v. at 8-h intervals). Treatment with bFF significantly (P less than 0.01) suppressed plasma FSH concentrations, but, at least for the first 30 h of treatment, did not influence the magnitude of GnRH-induced LH episodes (mean max. conc. 3.00 +/- 0.39 and 3.63 +/- 0.51 ng/ml for bFF-treated and control ewes, respectively). Of 10 animals treated with GnRH for 72 h, 5/5 control ewes showed oestrus and ovulated whereas 0/5 bFF-treated ewes showed oestrus or ovulated in response to GnRH treatment. There was, however, a transient (13.2 +/- 1.0 h) increase in plasma LH concentrations in the ewes given bFF (mean max. conc. 4.64 +/- 1.57 ng/ml), which was coincident with the preovulatory LH surge recorded in animals given GnRH alone. In 10 GnRH-treated ewes slaughtered after 32 h of treatment, the mean diameter of the largest antral follicle was significantly (P less than 0.001) greater in control ewes (5.92 +/- 0.17 mm) than in animals that were also given bFF (3.94 +/- 0.14 mm). In addition, the incidence of atresia in the 3 largest antral follicles present at this time was greater in bFF-treated ewes. These results show that, when plasma FSH concentrations are suppressed by administration of bFF, although the magnitude of GnRH-induced LH episodes is unchanged, preovulatory follicular development is impaired and ovulation does not occur.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacokinetic and pharmacodynamic interactions between simvastatin and diltiazem in patients with hypercholesterolemia and hypertension

Pharmacokinetic and pharmacodynamic interactions between simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, and diltiazem, a calcium antagonist, were investigated in 7 male and 4 female patients with hypercholesterolemia and hypertension. The patients were given, for one in a three consecutive 4-week periods, oral simvastatin (5 mg/day), oral simvastatin (5 mg/day) combined with diltiazem (90 mg/day), and then oral diltiazem (90 mg/day), respectively. The area under the plasma concentration versus time curve up to 6 hours post-dose (AUC0-6h) and maximum plasma concentrations (Cmax) of the drugs, serum lipid profiles, blood pressures and liver functions were assessed on the last day of each of the three 4-week periods. After the combined treatment period, Cmax of HMG-CoA reductase inhibitor was elevated from 7.8 +/- 2.6 ng/ml to 15.4 +/- 7.9 ng/ml (P < 0.01) and AUC0-6h from 21.7 +/- 4.9 ng x hr/ml to 43.3 +/- 23.4 ng x hr/ml (P < 0.01), while Cmax of diltiazem was decreased from 74.2 +/- 36.4 ng/ml to 58.6 +/- 18.9 ng/ml (P < 0.05) and its AUC0-6h from 365 +/- 153 ng x hr/ml to 287 +/- 113 ng x hr/ml (P < 0.01). Compared to simvastatin monotherapy, combined treatment further reduced LDL-cholesterol levels by 9%, from 129 +/- 16 mg/dl to 119 +/- 17 mg/dl (P < 0.05). No adverse events were observed throughout the study. These apparent pharmacokinetic interactions, namely the increase of HMG-CoA reductase inhibitor concentration by diltiazem and the decrease of diltiazem concentration by simvastatin, enhance the cholesterol-lowering effects of simvastatin during combined treatment.     

Determination of CGS 16617 and stable isotope-labeled CGS 16617, an angiotensin-converting enzyme inhibitor, in human plasma by gas chromatography/mass spectrometry

An analytical method has been developed for the simultaneous determination of a novel orally active angiotensin-converting enzyme inhibitor (CGS 16617) and a stable isotope-labeled analog. Both compounds are isolated from human plasma using an ion-exchange column, derivatized with pentafluoropropionic anhydride and pentafluoropropanol, and analyzed by gas chromatography/mass spectrometry. After splitless injection on a methyl-silicon column, the compound is detected using negative ion chemical ionization with nitrous oxide as a reagent gas. CGS 16617 labeled with four deuteriums and two 13C is used as an internal standard. The accuracy and precision of the method, expressed as the overall mean +/- SD recovery obtained from two sets of 36 quality-control samples used during a clinical study (concentration range 0.2-100 ng ml-1 plasma), was 96.1 +/- 16.2% for unlabeled drug and 97.6 +/- 14.4% for the D4-labeled drug (concentration range 0.2-100 ng ml-1 plasma). The limit of quantification using 1 ml plasma is 0.2 ng ml-1 for both labeled and unlabeled drug.     

Simultaneous determination of fixed dose combination of nebivolol and valsartan in human plasma by liquid chromatographic-tandem mass spectrometry and its application to pharmacokinetic study

A rapid, sensitive and accurate liquid chromatographic-tandem mass spectrometry method is described for the simultaneous determination of nebivolol and valsartan in human plasma. Nebivolol and valsartan were extracted from plasma using acetonitrile and separated on a C18 column. The mobile phase consisting of a mixture of acetonitrile and 0.05 mM formic acid (50:50 v/v, pH 3.5) was delivered at a flow rate of 0.25 ml/min. Atmospheric pressure ionization (API) source was operated in both positive and negative ion mode for nebivolol and valsartan, respectively. Selected reaction monitoring mode (SRM) using the transitions of m/z 406.1-->m/z 150.9; m/z 434.2-->m/z 179.0 and m/z 409.4-->m/z 228.1 were used to quantify nebivolol, valsartan and internal standard (IS), respectively. The linearity was obtained over the concentration range of 0.01-50.0 ng/ml and 1.0-2000.0 ng/ml and the lower limits of quantitation were 0.01 ng/ml and 1.0 ng/ml for nebivolol and valsartan, respectively. This method was successfully applied to the pharmacokinetic study of fixed dose combination (FDC) of nebivolol and valsartan formulation product after an oral administration to healthy human subjects.     

High performance liquid chromatographic-mass spectrometric determination of ginsenoside Rg3 and its metabolites in rat plasma using solid-phase extraction for pharmacokinetic studies

To support pharmacokinetic studies of ginsenosides, a novel method to quantitatively analyze ginsenoside Rg3 (Rg3), its prosapogenin ginsenoside Rh2 (Rh2) and aglycone 20(S)-protopanaxadiol (ppd) in rat plasma was developed and validated. The method was based on gradient separation of ginsenosides present in rat plasma using high performance liquid chromatography (HPLC), followed by detection with electrospray ionization(ESI) mass spectrometry (MS) in negative ion mode with the mobile phase additive, ammonium chloride (500 microM). Differentiation of ginsenosides was achieved through simultaneous detection of the [M(+)Cl(-)] adduct of ginsenoside Rg3 and [M(+)Cl(-)] adducts of its deglycosylated metabolites Rh2 and ppd, and other ions after solid phase extraction (SPE). The /specific ions monitored were m/z 819.50 for Rg3, m/z 657.35 for Rh2, m/z 495.40 for ppd and m/z 799.55 for the internal standard (digitoxin). The mean recoveries for Rg3, Rh2 and ppd were 77.85, 82.65 and 98.33%, respectively using 0.1 ml plasma for extraction. The lower limits of quantification were 10.0, 2.0 and 8.0 ng/ml (equivalent to 0.1, 0.02 and 0.08 ng in each 10 microl injection onto the HPLC column) for Rg3, Rh2 and ppd, respectively. The method has been demonstrated to be highly sensitive and accurate for the determination of Rg3 and its metabolites in rat plasma.     

Determination of zolmitriptan in human plasma by liquid chromatography-tandem mass spectrometry method: application to a pharmacokinetic study

A sensitive and selective liquid chromatography-tandem spectrometry method for the determination of zolmitriptan was developed and validated over the linearity range 0.05-30 ng/ml with 0.5 ml of plasma using diphenhydramine as the internal standard. Liquid-liquid extraction using a mixture of diethyl ether and dichloromethane was used to extract the drug and the internal standard from plasma. The mass spectrometer was operated under the selected reaction monitoring (SRM) mode using the atmospheric pressure chemical ionization (APCI) technique. The instrument parameters were optimized to obtain 3.0 min run time. The mobile phase consisted of acetonitrile-water-formic acid (70:30:0.5), at a flow rate of 0.5 ml/min. In positive mode, zolmitriptan produced a protonated precursor ion at m/z 288 and a corresponding product ion at m/z 58. And internal standard produced a protonated precursor ion at m/z 256 and a corresponding product ion at m/z 167. The inter- and intra-day precision (%R.S.D.) were less than 8.5% and accuracy (%error) was less than -2.5%. The method had a lower limit of quantification of 0.05 ng/ml for zolmitriptan, which offered increased sensitivity and selectivity of analysis, compared with existing methods. The method was successfully applied to a pharmacokinetic study of zolmitriptan after an oral administration of 5 mg zolmitriptan to 20 healthy volunteers.     

Relaxin regulates oxytocin secretion in late-pregnant beef heifers

The effects of porcine relaxin (3000 units/mg) on oxytocin (OT) and progesterone secretion were studied in beef heifers on Day 274 (10 days before expected parturition). Heifers (n = 11) were randomly assigned to three treatments: relaxin iv infusions combined with im injection (RLX-INF, 9000 units), relaxin im injection (RLX-im, 6000 units), and phosphate-buffered saline-treated controls (PBS). RLX-INF heifers received infusions of PBS and 1000 units of relaxin for 165 min, followed by 2000 units of relaxin im and finally 2000 units of relaxin infusion followed by 4000 units of relaxin im. Endogenous relaxin (immunoreactive) in the PBS-treated group was 0.2-0.9 ng/ml peripheral plasma. For the RLX-im group, peak relaxin was 81 +/- 12 ng/ml (+/- SE) at 45 min after treatment. There were two peaks of relaxin, 18 +/- 5.3 ng/ml and 74 +/- 7.5 ng/ml, 3.5-4.5 hr apart in the RLX-INF group. Significant peak releases of OT were evident in the relaxin-treated heifers. For the RLX-im group, an OT peak (42 +/- 16 pg/ml) occurred within 30 min after relaxin treatment. For the RLX-INF heifers, 2000 and 4000 units of relaxin were associated with major peaks of 14 +/- 0.5 and 43 +/- 1.7 pg/ml OT, respectively. Basal OT plasma levels in the PBS group were 2.5-3.1 pg/ml. Mean plasma progesterone for all heifers was 6.2 +/- 2.11 ng/ml before treatment. There was a significant decrease in progesterone (-2.5 ng/ml) in the RLX-im group within 60 min after relaxin treatment and 45 min after peak OT secretion. The maximum decrease in progesterone (-3.2 +/- 0.68 ng/ml) occurred 135 min after treatment in the RLX-im group. In the RLX-INF group, 2000 units of relaxin infusion combined with 4000 units of relaxin im significantly decreased progesterone (-3.2 +/- 1.59 ng/ml) in peripheral plasma. These results clearly indicate that relaxin causes an acute peak release of oxytocin within 30 min, followed by a marked decrease in plasma progesterone concentration in late-pregnancy cattle.     

Effect of FSH on ovarian inhibin secretion in anoestrous ewes

In Exp. 1, 7 Finn-Merino ewes which had one ovary autotransplanted to a site in the neck had jugular and timed ovarian venous blood samples collected at 10-min intervals for 2 h before and 3 h after injection of 5 micrograms NIAMDD-oFSH-S16. In Exp. 2, 8 Finn-Merino ewes with ovarian autotransplants had jugular and timed ovarian venous blood samples collected at 15-min intervals for 2 h before and 12 h after bolus injection of 40 micrograms NIAMDD-oFSH-S16 and infusion of oFSH-S16 at 6 micrograms/min for 4 h. In Exp. 2 the follicular population of the ovary was assessed by real-time ultrasound at the beginning and end of the experimental period. In both experiments the secretion rates of inhibin (1-3 ng/min) and oestradiol (0.5-8 ng/min) were similar to those observed during the luteal phase of the cycle in the breeding season, indicating significant follicular development in these animals. In Exp. 1 there was no change in the secretion of oestradiol or inhibin after the injection of FSH which resulted in a 25% increase (P less than 0.05) in the concentration of FSH in plasma. Inhibin secretion was pulsatile but there was no difference in inhibin pulse frequency before (1.6 +/- 0.2 pulses/h) or after (1.2 +/- 0.5 pulses/h) injection of FSH. In Exp. 2 injection of FSH resulted in an increase (P less than 0.001) in plasma concentrations of FSH in the sample taken 10 min after injection from a baseline of 1.2 +/- 0.2 ng/ml to a peak of 10.6 +/- 1.0 ng/ml (mean +/- s.e.m.).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of relaxin on facilitation of parturition in dairy heifers

Purified pig relaxin (3000 U/mg) was injected i.m. into pregnant Holstein dairy heifers on Day 276 or 277 to determine its effect on parturition and sequential measurements of the pelvic area, cervical dilatation, and peripheral blood-plasma concentrations of progesterone and relaxin. Treatments included phosphate-buffer saline (2 ml, Group C, N = 7), relaxin once (1 mg, Group 1R, N = 7), and twice (2 mg, 12 h apart; Group 2R, N = 7). Intervals (mean +/- s.e.) between the first injection of relaxin or PBS and calving were 64 +/- 17, 80 +/- 19 and 125 +/- 34 h for Groups 2R, 1R and C, respectively. The calving intervals were reduced in Groups 2R (P less than 0.01) and 1R (P less than 0.05) compared with Group C. The incidence of dystocia was 29% (2 of 7) in Group 2R and 43% (3 of 7) in Group 1R compared with 57% (4 of 7) in Group C (P less than 0.01). Body weights and ratios of males to females of the calves were similar (P greater than 0.05) between groups. Progesterone plasma concentrations decreased (P less than 0.01) earlier in Groups 1R and 2R compared with Group C, and this acute decrease began within 6 h of treatment. At 24 h after relaxin or PBS injection, progesterone concentrations were 2.7 +/- 1.1 ng/ml for Group 2R, 3.5 +/- 0.9 ng/ml for Group 1R, and 6.0 +/- 0.1 ng/ml for Group C. Relaxin reached peak blood-plasma levels of 19 +/- 2.2 ng/ml 1 h after injection of relaxin, but remained unchanged, 0.3 +/- 0.01 ng/ml, in Group C. Pelvic area was increased 26%, 22% and 14% and cervical dilatation was increased 109%, 76% and 53% 48 h after injection in Groups 2R, 1R and C, respectively, but these responses were similar among groups at the time of parturition. We conclude that two i.m. injections of relaxin facilitated earlier calving, acutely decreased progesterone secretion, increased cervical dilatation and pelvic area expansion, and decreased the incidence of dystocia in dairy heifers.     

Plasma growth hormone, insulin-like growth factor-I, and milk production responses to exogenous human growth hormone-releasing factor analogs in dairy cows

Responses of plasma growth hormone (GH) and insulin-like growth factor-I (IGF-I), and milk production to subcutaneous (sc) injection(s) of two synthetic human growth hormone-releasing factor (hGRF) analogs were studied in dairy cows. Two mg of each hGRF analog dissolved in 5 ml saline per cow were injected into the shoulder area of each experimental animal, and jugular venous blood samples were collected via an indwelling catheter or by venipuncture. Plasma GH and IGF-I concentrations were measured by radioimmunoassay methods. In dry cows, the mean concentration of plasma GH after a single sc injection of hGRF analogs rose to 22.0-28.3 ng/ml at about 5 h from 1.4-1.7 ng/ml at 0 h (just before injection), and returned to the level before injection after 10-12 h. On the other hand, the plasma IGF-I began to increase after a lag of 4-6 h following a single injection of hGRF analogs, and reached maximum values of 71.1-89.4 ng/ml at 20 h from 43.7-46.4 ng/ml at 0 h. The IGF-I concentration at 24 h after a single injection of hGRF analogs was still higher than the value for the dry cows given saline. In lactating cows, the plasma concentration of GH at 2 h after daily sc injections of hGRF analogs during 14 consecutive days (an injection period) was higher than those for the lactating cows which received saline. Also, during the injection period, the concentration of IGF-I was higher in the lactating cows which received hGRF analog injections than in the cows which received saline injections. During the last 7 days of the injection period, the administration of hGRF analogs increased the mean milk yield by 11-19% in comparison with those for the saline injected cows. A positive correlation was observed between the mean plasma IGF-I concentration and the mean milk yield in the lactating cows treated with hGRF analogs throughout the injection and a postinjection (11 consecutive days after cessation of hGRF analog injection) periods. The results demonstrate that a single sc injection of hGRF analogs stimulates both GH release and the circulating level of IGF-I in dry cows, and that daily sc injections of hGRF analogs over 14 days enhance milk production, and plasma GH and IGF-I levels in lactating cows.     

Rapid determination of omeprazole in human plasma by protein precipitation and liquid chromatography-tandem mass spectrometry

A rapid, sensitive and reliable method was developed to quantitate omeprazole in human plasma using liquid chromatography-tandem mass spectrometry. The assay is based on protein precipitation with acetonitrile and reversed-phase liquid chromatography performed on an octadecylsilica column (55 mm x 2mm, 3 microm particles), the mobile phase consisted of methanol-10 mM ammonium acetate (60:40, v/v). Omeprazole and flunitrazepam, the internal standard, elute at 0.80+/-0.10 min with a total run time 1.35 min. Quantification was through positive ion mode and selected reaction monitoring mode at m/z 346.1-->197.9 for omeprazole and m/z 314.0-->268.0 for flunitrazepam, respectively. The lower limit of quantitation was 1.2 ng/ml using 0.25 ml of plasma and linearity was observed from 1.2 to 1200 ng/ml. Within-day and between-day precision expressed by relative standard deviation was less than 5% and inaccuracy did not exceed 12%. The assay was applied to the analysis of samples from a pharmacokinetic study.