Huaibing He

Determination of catecholamines in sheep plasma by high-performance liquid chromatography with electrochemical detection: comparison of deoxyepinephrine and 3,4-dihydroxybenzylamine as internal standard.

3,4-Dihydroxybenzylamine (DHBA) is commonly used as the internal standard in HPLC catecholamine assays. Sheep are frequently used in studies of cardiovascular physiology and in such studies measurement of catecholamines is important. The recovery of DHBA from sheep plasma is, however variable and poor. Therefore, we have developed a reliable and sensitive HPLC-ED method with alumina extraction for measurement of catecholamines in sheep plasma using deoxyepinephrine (epinine) as the internal standard. Separation was performed on a muBondapak C18 column (300x3.9 mm, 10 microm) with a mobile phase containing 2% acetonitrile and 98% buffer (0.05% sodium acetate-0.02% EDTA-0.013% sodium heptanesulfonate), pH 3.25. The extraction of epinine from water, human plasma, dog plasma and sheep plasma did not differ (p>0.05), but extraction of DHBA from sheep plasma was significantly impaired (p<0.0001). The R2 of regression curves (n=5) of norepinephrine (NE) (25.02 pg/ml-1.00 ng/ml) and epinephrine (E) (25.82 pg/ml-1.03 ng/ml), using epinine as internal standard were greater than 0.99. The intra- and inter-day coefficients of variation were 2.11-11.15 and 0.88-12.60% for NE and 1.12-10.91 and 2.88-12.60% for E, respectively. The detection limits for NE and E are 12 pg/ml. The technique described has the advantage that it allows the simultaneous determination of both endogenous and [3H]norepinephrine in sheep plasma using a sensitive and reproducible HPLC technique.

Optimization of high-performance liquid chromatographic assay for catecholamines: Determination of optimal mobile phase composition and elimination of species-dependent differences in extraction recovery of 3,4-dihydroxybenzylamine

This paper describes the application of a window diagram technique to optimize the four components of eluent (sodium acetate, sodium heptanesulfonate, acetonitrile and pH adjusted by monochloroacetic acid), for complete separation of five catecholamine compounds and the internal standard (3,4-dihydroxybenzylamine, DHBA). In addition, studies were performed to address the problem of the variable recovery of DHBA from dog plasma due to a time-dependent loss of DHBA. We found that this phenomenon can be prevented by pH adjustment prior to addition of DHBA, allowing development of an accurate high-performance liquid chromatographic assay for plasma catecholamines in dogs.

β‐Adrenergic receptor—mediated release of norepinephrine in the human forearm

Because of difficulties in separating the systemic from local effect, the role of presynaptic β2‐adrenergic receptors in facilitating the neural release of norepinephrine has not been defined previously in humans in vivo. To determine whether stimulation of presynaptic β‐receptors alters local release of norepinephrine, we examined the effects on norepinephrine kinetics of 60 and 400 ng/min intra‐arterial isoproterenol in seven healthy male volunteers. Isoproterenol, 60 ng/min, increased forearm norepinephrine spillover sixfold from a baseline spillover of 0.45 ± 0.08 to 2.89 ± 0.69 ng/min (p < 0.01), whereas 400 ng/min isoproterenol increased forearm norepinephrine spillover to 13.25 ± 2.49 ng/min (p < 0.005), a 29‐fold increase above baseline. Coinfusion of 20 to 40 µg/min propranolol with 400 ng/min isoproterenol in four subjects significantly attenuated the isoproterenol‐induced increase in local norepinephrine spillover to 2.09 ± 0.92 ng/min (p < 0.05 versus 400 ng/min isoproterenol). This study shows that presynaptic ß‐adrenergic receptors facilitate local release of norepinephrine in vivo in humans.

Simultaneous determination of codeine and it seven metabolites in plasma and urine by high-performance liquid chromatography with ultraviolet and electrochemical detection

A sensitive and selective high-performance liquid chromatography method has been developed for the measurement of codeine and its seven metabolites, norcodeine, morphine, normorphine, codeine-6-glucuronide, morphine-6-glucuronide, morphine-3-glucuronide and norcodeine glucuronide, in plasma and urine. The compounds were recovered from plasma and urine using solid-phase extraction with C18 cartridges and separated on a reversed-phase C8 column with a mobile phase consisting of 77% buffer (5 mM sodium phosphate monobasic and 0.70 mM sodium dodecyl sulfate, pH 2.35) and 23% acetonitrile. Codeine, norcodeine, codeine-6-glucuronide, norcodeine glucuronide and morphine-3-glucuronide were detected by ultraviolet detection at 214 nm, with a detection limit of 0.02 nmol/ml for each compound in plasma. Morphine-6-glucuronide, normorphine and morphine were monitored by electrochemical detection at 350 mV, with a detection limit of 0.003 nmol/ml for each compound in plasma. The assay showed good reproducibility and accuracy using external standardization. The recovery and inter-day variation for all compounds in plasma samples were 63.40-77.90% and 3.49-16.77% (R.S.D.) and while in urine were 64.98-90.13% and 2.93-9.96% (R.S.D.), respectively.

Blunted Blood Pressure Response to Central Sympathoinhibition in Normotensive Blacks: Increased Importance of Nonsympathetic Factors in Blood Pressure Maintenance in Blacks

Enhanced sympathetic reactivity may predispose blacks to the development of hypertension and may occur because of increased sympathetic stimulation and/or attenuated sympathoinhibition. A potential site for such attenuated sympathetic inhibition may be at the level of central alpha2-adrenergic receptors, which play an important role in the feedback inhibition of norepinephrine release. We used cumulative doses (1, 2, and 3 microg/kg I.V.) of the centrally acting alpha2-adrenergic agonist clonidine to measure the sensitivity of alpha2-adrenoceptor-mediated sympathoinhibition and the resultant hypotensive response in 8 normotensive blacks and 10 normotensive whites. Sympathetic activity was determined by radioisotope dilution methodology. Basal norepinephrine spillover was similar in blacks (0.80+/-0.14 microg/min) and whites (0.73+/-0.19 microg/min, P=NS) and after clonidine decreased significantly in both blacks (0.21+/-0.07 microg/min, P<.0001) and whites (0.24+/-0.06 microg/min, P<.0001), with no difference between the groups (P=NS). Despite this similar degree of sympathoinhibition, the hypotensive response to clonidine was markedly blunted in blacks, such that mean arterial pressure decreased by only 10% in blacks but by 21% in whites (P<.0001). The smaller blood pressure decrement after clonidine in normotensive blacks, in the face of an equal degree of sympathoinhibition, suggests that even when sympathetic tone is decreased to the same level in blacks and whites, normotensive blacks have less reduction in blood pressure than whites, implying that nonadrenergic mechanisms contribute more to blood pressure maintenance in blacks than whites. Whether a similar interethnic difference in response to sympathoinhibition occurs in hypertensive patients is as yet unknown.

Quantitation of loperamide and N-demethyl-loperamide in human plasma using electrospray ionization with selected reaction ion monitoring liquid chromatography-mass spectrometry

We report here the development and validation of an LC-MS method for quantitation of loperamide (LOP) and its N-demethyl metabolite (DMLOP) in human plasma. O-Acetyl-loperamide (A-LOP) was synthesized by us for use as an internal standard in the assay. After addition of the internal standard, the compounds of interest were extracted with methyl tert.-butylether and separated by HPLC on a C18 reversed-phase column using an acetonitrile-water gradient containing 20 mM ammonium acetate. The three compounds were well separated by HPLC and no interfering peaks were detected at the usual concentrations found in plasma. Analytes were quantitated using positive electrospray ionization in a triple quadrupole mass spectrometer operating in the MS-MS mode. Selected reaction monitoring was used to quantify LOP (m/z 477-266), DMLOP (m/z 463-->252) and A-LOP (m/z 519-->266) on ions formed by loss of the 4-(p-chlorophenyl)-4-hydroxy-piperidyl group upon low energy collision-induced dissociation. Calibration curves, which were linear over the range 1.04 to 41.7 pmol/ml (LOP) and 1.55 to 41.9 pmol/ml (DMLOP), were run contemporaneously with each batch of samples, along with low (4.2 pmol/ml), medium (16.7 pmol/ml) and high (33.4 pmol/ml) quality control samples. The lower limit of quantitation (LLQ) of LOP and DMLOP was about 0.25 pmol/ml in plasma. The extraction efficiency of LOP and DMLOP from human plasma was 72.3+/-1.50% (range: 70.7-73.7%) and 79.4+/-12.8% (64.9-88.8%), respectively. The intra- and inter-assay variability of LOP and DMLOP ranged from 2.1 to 14.5% for the low, medium and high quality control samples. The method has been used successfully to study loperamide pharmacokinetics in adult humans.

Determination of low plasma timolol concentrations following topical application of timolol eye drops in humans by high-performance liquid chromatography with electrochemical detection

A simple and sensitive high-performance liquid chromatographic assay was developed for determination of timolol in human plasma following administration of two drops of a 5% timolol ophthalmic solution. A 4% butyl alcohol-hexane extract of an alkalized sample of plasma was chromatographed on a reversed-phase column and the components in the column effluent were monitored by coulometric detection. The extraction efficiency of timolol was 69.02 +/- 4.16% (mean +/- S.D.) and its detection limit was 107.2 pg/ml. The effect of mobile phase pH, buffer concentration and the working potential of the detector on column performance and the electrochemical response are described.

Norepinephrine Release in the Human Forearm: Effects of Epinephrine

It has been postulated that delayed facilitation of norepinephrine release by epinephrine is causally related to the development of hypertension. It has been proposed that a brief increase in epinephrine concentrations results in the uptake of epinephrine into the sympathetic nerve terminal. Subsequent rerelease of epinephrine stimulates presynaptic beta-adrenergic receptors, resulting in a prolonged increase in plasma norepinephrine (NE) concentrations, with amplified sympathetic responses and vasoconstriction. To determine whether such epinephrine-induced, delayed facilitation of NE release occurs in a vascular bed draining resistance vessels and, if it occurs, whether that facilitation differs in hypertension, we used a radioisotope dilution method to measure unstimulated and isoproterenol-stimulated forearm NE spillover before, during, and after a 50 ng/min infusion of epinephrine for 30 minutes directly into the brachial artery. No delayed facilitatory effects of epinephrine on forearm NE spillover were observed in either 6 normotensive (NT) or 8 borderline hypertensive (BHT) subjects (NT unstimulated forearm NE spillover preepinephrine 1.79+/-0.41 ng/min versus postepinephrine 2.36+/-0.65 ng/min, P=.38; BHT preepinephrine 2.24+/-0.70 ng/min versus postepinephrine 1.93+/-0.46 ng/min, P=.51; NT isoproterenol-stimulated forearm NE spillover preepinephrine 4.61+/-1.01 ng/min versus postepinephrine 4.4+/-0.98 ng/min, P=.9; BHT preepinephrine 4.04+/-1.36 ng/min versus postepinephrine 4.69+/-1.49 ng/min P=.5). We conclude that the short-term local infusion of epinephrine does not have a delayed facilitatory effect on forearm NE spillover in NT or BHT subjects. Therefore, the prolonged increase in NE concentrations after epinephrine infusion previously shown systemically, and not seen locally in the forearm, suggests that the delayed facilitatory response to epinephrine may occur in other organs.

Tachyphylaxis of human forearm vascular responses does not occur rapidly after exposure to isoproterenol.

In vitro and limited in vivo data suggest that rapid desensitization of beta-adrenoceptor responses occurs after exposure to agonist. Tachyphylaxis to a beta-adrenoceptor agonist would represent a potentially important mechanism for the short-term regulation of vascular tone. The effects of a 4-hour infusion of 400 ng/min intra-arterial isoproterenol on forearm blood flow and presynaptic beta-adrenoceptor-mediated norepinephrine release were determined in eight healthy volunteers. Intra-arterial isoproterenol at 400 ng/min resulted in a significant increase in forearm blood flow in all eight subjects at all time points, with no evidence of tachyphylaxis. In fact, forearm blood flow after 4 hours of the isoproterenol infusion (22.8 +/- 3.3 mL/100 mL per minute) was significantly greater than after 7 minutes (14.6 +/- 2.8 mL/100 mL per minute), 15 minutes (15.4 +/- 2.4 mL/100 mL per minute), and 30 minutes (17.4 +/- 3.0 mL/100 mL per minute) of the infusion (P < .05). Similarly, presynaptic beta-adrenoceptor responses showed no evidence of tachyphylaxis, so forearm norepinephrine spillover values after 7 minutes (6.6 +/- 0.94 ng/min), 15 minutes (7.6 +/- 1.5 ng/min), and 4 hours (8.8 +/- 1.1 ng/min) of isoproterenol infusion were increased and similar. Minimal systemic effects were observed, and there was no evidence of tolerance, there being no difference in heart rate after 7 minutes (70.7 +/- 2.7 beats per minute) and 4 hours (72.2 +/- 3.6 beats per minute) of isoproterenol infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary sodium intake modulates systemic but not forearm norepinephrine release

Sodium intake has profound effects on systemic and renal sympathetic activity, but its effects on sympathetic activity in skeletal muscle vascular beds, a site at which local regulatory mechanisms could alter vascular tone directly, are unclear.