Rapid confirmation/quantitation of ecgonine methyl ester, benzoylecgonine, and cocaine in urine using on-line extraction coupled with fast HPLC and tandem mass spectrometry.

A rapid, rugged, and highly specific assay for the quantitation of cocaine (COC) and especially its primary metabolites benzoylecgonine (BZE) and ecgonine methyl ester (EME) in human urine has been established. Here, we investigated the use of on-line sample extraction coupled to rapid chromatography systems for tandem mass analysis of COC, EME, and BZE in human urine. Using this method, sample preparation consisted of a sole centrifugation step. Combined extraction and chromatographic run times were < 3.5 min. The lower limits of detection were 0.5 ng/mL, 2.0 ng/mL, and 0.5 ng/mL for EME, BZE, and COC, respectively. Linear calibration curves ranging from 7.5 ng/mL to 1000 ng/mL were produced for the test analytes. Within-day and between-day precision and accuracy of the assay were determined using human urine quality-control specimens at 5, 10, or 15; 150; and 1000 ng/mL. The analyses were performed over the course of five days, rendering %CVs < 10% for EME, BZE, and COC. Percent mean accuracy for the three analytes of 97 to 113% were obtained. Our data suggest that on-line sample extraction coupled with rapid high-performance liquid chromatography-tandem mass spectrometry may be a viable alternative for EME, BZE, and COC analyses in human urine.

Performance of a pentafluorophenylpropyl stationary phase for the electrospray ionization high-performance liquid chromatography-mass spectrometry-mass spectrometry assay of cocaine and its metabolite ecgonine methyl ester in human urine.

A pentafluorophenylpropyl (PFPP) bonded silica column has been used for the high-performance liquid chromatography-electrospray ionization-mass spectrometry-mass spectrometry assay (HPLC-ESI-MS-MS) of cocaine (COC) and its metabolite, ecgonine methyl ester (EME) in human urine. COC and EME were used as model basic solutes to demonstrate that a PFPP phase yields excellent results for the assay and validation of drugs in biological fluids. The assay was linear over three orders of magnitude (1.0-1000 ng/ml) and precision and accuracy of the assay was 4 and 15%, respectively. The limit of detection (LOD) for COC and EME was 1.6 and 2.8 pg on column, respectively. In addition, only a simple 1:10 dilution of the urine was necessary for the sample preparation procedure thus saving time on a laborious extraction step. The major advantage of the PFPP phase was the enhancement of the ESI-MS signal by providing good retention and good peak shape of COC and EME with a mobile phase of 90% acetonitrile. The MS signal for COC was a factor of 12 times greater on the PFPP phase than on the C18 phase.

Direct determination of ecgonine methyl ester and cocaine in rat plasma, utilizing on-line sample extraction coupled with rapid chromatography/quadrupole orthogonal acceleration time-of-flight detection.

Our current experiments assess the applicability of on-line sample extraction with coupled rapid chromatography systems to quadrupole orthogonal acceleration time-of-flight (Q-TOF) detection for the quantitative analysis of cocaine (COC), and ecgonine methyl ester (EME) in rat plasma. Experiments were performed on a Q-TOF instrument, operated in the MS/MS mode. Quantitation was achieved utilizing the most prominent parent-daughter transition and internal standard calibration techniques (COC-d3: IS). The calibration curves produced for EME and COC ranged from 5.0 to 10,000 and 0.5 to 10,000 ng/ml, respectively. Equations of regression line and correlation coefficients for the pseudo-multiple reaction monitoring (MRM) ion abundance ratio and the corresponding calibration concentrations (r2) were as follows: y = 0.0003 + 0.0703x (r2 = 0.9921) for EME and y = 0.0032 + 0.0035x (r2 = 0.9997) for COC. The system repeatability, given as percent coefficient of variation (% CV) of mean peak-area ratios, was assessed using 50 injections of a rat plasma sample from the pharmacokinetic study. The analyses were performed over the course of 5 days, rendering % CVs for EME and COC of 0.73 and 0.58, respectively. This method suggests that on-line sample extraction coupled with fast liquid chromatography/quadrupole orthogonal time-of-flight mass spectrometry may be a viable alternative for quantitative analysis of EME and COC in rat plasma.

Rapid confirmation/quantitation of cocaine and benzoylecgonine in urine utilizing high performance liquid chromatography and tandem mass spectrometry.

A rapid, but sensitive and selective method for confirmation and quantitation of benzoylecgonine (BZE) and cocaine (COC) in urine by fast-gradient liquid chromatography/tandem mass spectrometry (LC/MS/MS) is described. The chromatographic separation was performed on a reversed phase column employing fast-gradient techniques. Matrix prepared standards, blanks, and QC's were filtered then aliquots were transferred into a 96 well plate. Injection volumes of 25 microL were made onto the analytical column, with the flow diverted from the atmospheric pressure ionization source for the first 0.5 min of the analysis. Simultaneous multiple reaction monitoring (MRM) of three discrete transitions for each compound were used to identify BZE and COC. Quantitation was achieved utilizing the most prominent parent-daughter transition and internal standard calibration techniques. The coefficients of variation (CV) for the analysis of these drugs ranged from 0.6% to 6.8% at a concentration of 150 ng/mL (n = 155). This method suggests that fast-gradient LC/MS/MS may be suitable for routine confirmation of immunoassay cocaine-positive samples.

Altered lens short-circuit current in adult cataract-prone Dahl hypertensive rats.

We assessed components of lenticular short-circuit current in adult hypertensive Dahl salt-sensitive rats (DS) during chronic control (0.4% sodium) versus high (3% sodium) dietary NaCl intake begun at the age of 4 weeks until rats were studied. We also evaluated the influence of barium, a potassium channel blocker, and ouabain, a specific inhibitor of Na+, K(+)-ATPase activity, by adding them to the anterior lens surface, thus measuring barium-sensitive, ouabain-sensitive, and barium- and ouabain-in-sensitive short-circuit currents. During control NaCl intake, short-circuit current in DS and their control group, Dahl salt-resistant rats (DR), did not differ significantly. DS were subclassified into cataract-prone rats and rats unlikely to develop cataracts on the basis of their initial pressor response to the change from a normal to high NaCl diet during the first weeks of age. Although only transparent lenses were studied, total lens short-circuit current was already markedly decreased in the cataract-prone subgroup compared with DS unlikely to develop cataracts and control DR. This was in sharp contrast to the increase in short-circuit current previously reported in Sprague-Dawley rats and now observed in control DR in response to high dietary NaCl. The decrease in lens short-circuit current in cataract-prone rats was associated with lower absolute values of barium- and ouabain-sensitive short-circuit currents as well as with low barium- and ouabain-insensitive short-circuit current. Although the barium- and ouabain-sensitive components of the short-circuit current were similar in DS unlikely to develop cataracts and DR, the barium- and ouabain-insensitive component of the short-circuit current was lower in DS unlikely to develop cataracts than values in DR. Interestingly, this component of lens short-circuit current also increased in DR during chronic high NaCl, whereas the opposite change occurred in cataract-prone DS and DS unlikely to develop cataracts. Thus, the barium- and ouabain-insensitive short-circuit current may be a mechanism that protects the normal lens from developing cataracts. Possible candidates for this short-circuit current component are voltage-dependent potassium channels, calcium-activated potassium channels, or both. Our studies show altered lens short-circuit current in response to high NaCl intake in cataract-prone DS and suggest the possibility of altered lens potassium transport during sustained hypertension but before loss of lens transparency.

Increased dietary NaCl intake influences lens transport properties in Sprague-Dawley rats.

Several years ago, we reported a high frequency of cataracts in Dahl salt-sensitive rats (DS) which led us to carry out a series of studies, such as longitudinal ouabain-sensitive lens 86Rb uptake measurements. Yet, throughout all of our work, we used the Dahl salt-resistant rat (DR) as our control group, despite the fact that DR is a strain resistant to even experimental hypertension such as that resulting from renal cellophane wrapping. Thus, in the present study we assessed lens short-circuit current (Isc) in adult Sprague-Dawley rats (SD) from which DS and DR were derived. Sprague-Dawley rats were studied during chronic normal vs high NaCl intake. These studies showed that basal Isc and calculated translenticular potential difference (PDt) were nearly doubled by this dietary regimen. Sequential addition of BaCl2 and ouabain to the isolated lens anterior surface bath also revealed increased BaCl2-sensitive as well as BaCl2- and ouabain-insensitive lens Isc in SD kept on a high NaCl diet. There was a statistically insignificant tendency for lens ouabain-sensitive Isc to increase in SD given a high NaCl diet. These findings were unrelated to levels of arterial blood pressure which were not altered by chronic high NaCl intake in adult SD. Our data suggest the possibility of increased lens barium and ouabain insensitive ionic permeabilities combined with possibly increased lens fiber potassium concentration, as a response to chronic high dietary NaCl in the normal rat. The nature of the barium and ouabain insensitive component of the lens Isc that rises during chronic high NaCl intake requires elucidation.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of active and passive Na+ and K+ transport in normal rat lens by the short-circuiting technique.

An initial characterization of the lenticular ionic permeabilities of the isolated Sprague-Dawley rat lens utilizing short-circuiting techniques was carried out to provide the basis for further studies of mechanisms underlying cataractogenesis associated with salt-sensitive genetic hypertension in the rat. Both active and passive Na+ and K+ transport were evaluated by varying ionic concentrations in the bathing solutions facing the anterior and posterior sides of the lens, as well as by the addition of BaCl2 and ouabain. In general, the ionic permeabilities and transport properties of the rat lens are qualitatively similar to those previously described in other species. Ionic replacement studies showed the presence of Na+ and K+ channels at both surfaces of the lens, with the anterior side K+ conductance being larger than the posterior. In contrast, Na+ conductance was similar at both lens surfaces. The effects of ouabain confirmed the presence of the Na(+)-K(+)-ATPase at the lens epithelium, while the effects of serial addition of BaCl2 and ouabain suggested that the contribution of K+ diffusion to the short-circuit current may be considerably greater than the electrogenic component of the Na(+)-K+ pump.

Lenticular rubidium uptake and plasma renin activity in weanling cataract-prone salt-sensitive rats.

Our earlier studies of cataracts in Dahl salt-sensitive (DS) rats suggested the possibility of altered lens ion transport as a contributing factor in cataractogenesis in this genetic model. We also observed that those weanling DS rats with the greatest pressor response to a high salt diet eventually developed cataracts, and that changes in salt intake modified cataract formation. In the present studies, we measured lens 86Rb uptake as an index of sodium-potassium adenosine triphosphatase [(Na+,K+)-ATPase] activity in weanling DS rats before the development of cataracts or sustained hypertension. Additionally, plasma renin activity was measured to indirectly assess our hypothesis that the difference between cataract-prone DS rats and DS rats unlikely to develop cataracts might be a difference in degree of salt sensitivity. At the age of 4 weeks, 50 DS and 25 salt-resistant (DR) rats were given a high sodium diet for 2 weeks, at which time the rats were divided into three groups based on the systolic blood pressure response, that is, cataract-prone DS rats with systolic blood pressure equal to or greater than 155 mm Hg, DS rats unlikely to develop cataracts with systolic blood pressure less than or equal to 125 mm Hg, and DR rats. Lens and aqueous humor Na+ and K+, lens dry weight, and water content were not significantly different among the three groups of weanling rats. Plasma renin activity was lowest in cataract-prone DS rats and low in DS rats unlikely to develop cataracts when compared with values in DR rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention and reversal of cataracts in genetically hypertensive rats through sodium restriction.

We previously described the Dahl salt-sensitive rat as a potential model of cataractogenesis in which cataract formation is associated with hypertension. Cataractous lesions were characterized by a marked lenticular and aqueous humor electrolyte imbalance. In the present study the effects of chronic dietary sodium restriction on cataract formation were evaluated in salt-sensitive rats to determine whether or not modification of the hypertensive process might reduce the incidence of cataracts in this genetic model. In addition, the possibility that early cataractous lesions in adult hypertensive salt-sensitive rats might be reversed by acute sodium restriction was evaluated. Chronic dietary sodium restriction modified the development of hypertension and prevented cataract formation in salt-sensitive rats. Furthermore, acute dietary sodium restriction (1 week) completely and consistently reversed early cataractous lesions (pinpoint opacities) in adult hypertensive salt-sensitive rats. Both the prevention and reversal of cataracts were associated with normalization of the lenticular and aqueous humor parameters measured. These data suggest that cataractogenesis is not the consequence of sustained arterial hypertension, but rather that initiation of both hypertension and cataract formation in this genetic model may be the result of extracellular fluid volume state.