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A method for the simultaneous determination of dextromethorphan (DEX), dextrorphan (DET), and guaifenesin (GG) in human plasma was developed, validated, and applied to determine plasma concentrations of these compounds in samples from six clinical pharmacokinetic (PK) studies. Semi-automated liquid handling systems were used to perform the majority of the sample manipulation including liquid/liquid extraction (LLE) of the analytes from human plasma. Stable-isotope-labeled analogues were utilized as internal standards (ISTDs) for each analyte to facilitate accurate and precise quantification. Extracts were analyzed using gradient liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Use of semi-automated LLE with LC-MS/MS proved to be a very rugged and reliable approach for analysis of more than 6200 clinical study samples. The lower limit of quantification was validated at 0.010, 0.010, and 1.0 ng/mL of plasma for DEX, DET, and GG, respectively. Accuracy and precision of quality control (QC) samples for all three analytes met FDA Guidance criteria of +/-15% for average QC accuracy with coefficients of variation less than 15%. Data from the thorough evaluation of the method during development, validation, and application are presented to characterize selectivity, linearity, over-range sample analysis, accuracy, precision, autosampler carry-over, ruggedness, extraction efficiency, ionization suppression, and stability. Pharmacokinetic data are also provided to illustrate improvements in systemic drug and metabolite concentration-time profiles that were achieved by formulation optimization.

An on-line liquid chromatography/tandem mass spectrometry (LC-MS/MS) procedure, using the Prospekt- 2 system, was developed and used for the determination of the levels of the active ingredients of cough/cold medications in human plasma matrix. The experimental configuration allows direct plasma injection by performing on- line solid phase extraction (SPE) on small cartridge columns prior to elution of the analyte(s) onto the analytical column and subsequent MS/MS detection. The quantitative analysis of three analytes with differing polarities, dextromethorphan (DEX), dextrorphan (DET) and guaifenesin (GG) in human plasma presented a significant challenge. Using stable-isotope-labeled internal standards for each analyte, the Prospekt-2 on-line methodology was evaluated for sensitivity, suppression, accuracy, precision, linearity, analyst time, analysis time, cost, carryover and ease of use. The lower limit of quantitation for the on-line SPE procedure for DEX, DET and GG was 0.05, 0.05 and 5.0 ng mL(-1), respectively, using a 0.1 mL sample volume. The linear range for DEX and DET was 0.05-50 ng mL(-1) and was 5-5,000 ng mL(-1) for GG. Accuracy and precision data for five different levels of QC samples were collected over three separate days. Accuracy ranged from 90% to 112% for all three analytes, while the precision, as measured by the %RSD, ranged from 1.5% to 16.0%

A semi-automated, 96-well based liquid-liquid back-extraction (LLE) procedure was developed and used for sample preparation of dextromethorphan (DEX), an active ingredient in many over-the-counter cough formulations, and dextrorphan (DOR), an active metabolite of DEX, in human plasma. The plasma extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The analytes were isolated from human plasma using an initial ether extraction, followed by a back extraction from the ether into a small volume of acidified water. The acidified water isolated from the back extraction was analyzed directly by LC-MS-MS, eliminating the need for a dry down step. A liquid handling system was utilized for all aspects of liquid transfers during the LLE procedure including the transfer of samples from individual tubes into a 96-well format, preparation of standards, addition of internal standard and the addition and transfer of the extraction solvents. The semi-automated, 96-well based LLE procedure reduced sample preparation time by a factor of four versus a comparable manually performed LLE procedure.

The utility of packed-column supercritical, subcritical, and enhanced fluidity liquid chromatographies (pcSFC) for high-throughput applications has increased during the past few years. In contrast to traditional reversed-phase liquid chromatography, the addition of a volatile component to the mobile phase, such as CO2, produces a lower mobile-phase viscosity. This allows the use of higher flow rates which can translate into faster analysis times. In addition, the resulting mobile phase is considerably more volatile than the aqueous-based mobile phases that are typically used with LC-MS, allowing the entire effluent to be directed into the MS interface. High-throughput bioanalytical quantitation using pcSFC-MS/MS for pharmacokinetics applications is demonstrated in this report using dextromethorphan as a model compound. Plasma samples were prepared by automated liquid/liquid extraction in the 96-well format prior to pcSFC-MS/MS analysis. Three days of validation data are provided along with study sample data from a patient dosed with commercially available Vicks 44. Using pcSFC and MS/MS, dextromethorphan was quantified in 96-well plates at a rate of approximately 10 min/plate with average intraday accuracy of 9% or better. Daily relative standard deviations (RSDs) were less than 10% for the 2.21 and 14.8 ng/mL quality control (QC) samples, while the RSDs were less than 15% at the 0.554 ng/mL QC level.

The determination of dextromethorphan in canine plasma is used to demonstrate the high throughput bioanalytical approach of automated dilute-and-shoot (DAS) sample preparation followed by a 1 min isocratic liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Dilute-and-shoot preparation is commonly used for the determination of drugs in several biological matrices such as urine and saliva, but is not typically used with plasma samples because the amount of protein present in plasma can lead to a variety of problems including column failure. As a result, plasma sample preparation usually removes protein by precipitation, extraction or filtration; however, the dilute-and-shoot approach solubilizes proteins throughout the chromatographic portion of the assay. The attributes of this approach are compared with a previously validated liquid/liquid extraction procedure for determination of dextromethorphan in plasma. Accuracy and precision of both methods are similar. The lower limit of quantitation (LLOQ) of the dilute-and-shoot approach is much higher at 2 ng/ml versus 5 pg/ml with the liquid/liquid extraction; however, the sample throughput of the preparation portion of the dilute-and-shoot approach is more than 50-fold greater. The ruggedness of the dilute-and-shoot method was thoroughly investigated because of the problems traditionally associated with the direct injection of diluted plasma onto an LC-MS/MS instrument. With the optimal conditions, greater than 1,000 injections of diluted plasma have been successfully performed on a single column in less than 19 h making this technique an excellent approach for the rapid preparation and high throughput of plasma samples containing drug levels in the ng/ml range or higher. Application of this methodology to measure the levels of dextromethorphan in canine plasma to evaluate drug delivery from various formulations is also presented.

The popularity of packed-column supercritical fluid, subcritical fluid, and enhanced fluidity liquid chromatographies (pcSFC) for enantiomeric separations has increased steadily over the past few years. The addition of a significant amount (typically 20-95%) of a viscosity lowering agent, such as carbon dioxide, to the mobile phase provides a number of advantages for chiral separations. For example, higher mobile-phase flow rates can often be attained without a concomitant loss in chromatographic efficiency since diffusion coefficients, and optimum velocities, are typically higher in pcSFC. Ultratrace enantioselective quantitation of drugs in biomatrixes is an ideal application for these chromatographic attributes. To demonstrate the utility of this approach, a pcSFC tandem mass spectrometry (pcSFC-MS/MS) method was compared to a LC-MS/MS method for quantitation of the (R)- and (S)-enantiomers of ketoprofen (kt), a potent nonsteroidal, anti-inflammatory drug, in human plasma. After preparation using automated solid-phase extraction in the 96-well format, kt enantiomers were separated on a Chirex 3005 analytical column using isocratic conditions. Validation data and study sample data from patients dosed with either orally or topically administered ketoprofen were generated using both pcSFC and LC as the chromatographic methods to compare and contrast these analytical approaches. Generally, most analytical attributes, including specificity, linearity, sensitivity, accuracy, precision, and ruggedness, for both of these methods were comparable with the exception that the pcSFC separation provided a roughly 3-fold reduction in analysis time. A 2.3-min pcSFC separation and a 6.5-min LC separation provided equivalent, near-baseline-resolved peaks, demonstrating a significant time savings for analysis of large batch pharmacokinetic samples using pcSFC.

A sensitive and selective method was developed for the determination of (R)-ketoprofen ((R)-kt) and (S)-ketoprofen ((S)-kt) in human plasma using chiral liquid chromatography/tandem mass spectrometry (LC/MS/MS). Plasma samples spiked with stable-isotope-labeled [(13)C(1), (2)H(3)]-(R and S)-ketoprofen, for use as the internal standards, were prepared for analysis using automated solid-phase extraction (SPE) in the 96-well microtiter format. The enantiomers were separated on an (R)-1-naphthylglycine and 3,5-dinitrobenzoic acid (Chirex 3005) 250x2.0 mm i.d. analytical column, equipped with a 30x2.0 mm i.d. guard column using isocratic mobile phase conditions. The (R)- and (S)-kt levels were quantifiable from 0.05 to 2500 ng ml(-1) by constructing two separate curves from calibration standards covering the same range. The first curve ranged from 0.05 to 100 and the second from 100 to 2500 ng ml(-1). A concentration of 0.05 ng ml(-1) of either enantiomer was easily detected using a 1 ml plasma sample volume. The average method accuracy, evaluated at four levels over an extended period, was better than +/-3% over the entire range. The precision for the same set of quality control samples ranged from 4.0 to 7.0 % RSD (n = 24). The method was applied to the evaluation of pharmacokinetic parameters in human plasma obtained from volunteers who received 25 mg of kt by peroral administration of Actron caplets or by topical administration of Oruvail gel., (Copyright 2000 John Wiley & Sons, Ltd.)

A stable-isotope based gas chromatography-tandem mass spectrometry-negative ion chemical ionization method was developed for the determination of norepinephrine (NE) levels in small volumes (25-100 microl) of plasma. NE was stabilized in plasma by the addition of semicarbazide and spiked with deuterium-labeled norepinephrine internal standard. The analytes were isolated from the plasma by solid-phase extraction using phenylboronic acid columns and derivatized using pentafluoropropionic anhydride. The derivatized analytes were chromatographed on a capillary column and detected by tandem mass spectrometry with negative ion chemical ionization. Unparalleled sensitivity and selectivity were obtained using this detection scheme, allowing the unambiguous analysis of trace levels of NE in small-volume plasma samples. Linear standard curves were obtained for NE over a mass range from 1 to 200 pg per sample. The method had a limit of quantitation of 10 pg NE/ml plasma when using a 100-microl sample aliquot (1 pg/sample). Accuracy for the analysis of plasma samples spiked with 10 to 200 pg NE/ml typically ranged from 100+/-10%, with RSD values of less than 10%. The methodology was applied to determine the effect of clonidine on plasma NE levels in conscious spontaneously hypertensive rats. Administration of clonidine (30 microg/kg) produced an approximately 80% reduction in plasma NE accompanied by a 30% reduction in heart and mean arterial pressure that persisted >90 min after drug administration. The ability to take multiple samples from individual rats allowed the time course for the effect of clonidine to be mapped out using only one group of animals.

Rapid, sensitive and selective methods were developed for the determination of dextromethorphan and its major metabolite, dextrorphan, in human plasma using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Plasma samples spiked with stable-isotope internal standards were prepared for analysis by a liquid-liquid back-extraction procedure. Dextromethorphan and dextrorphan were chromatographed on a short reversed-phase column, using separate isocratic mobile phase conditions optimized to elute each compound in approximately 1.1 min. For both analytes, calibration curves were obtained over four orders of magnitude and the limit of quantitation was 5 pg ml-1 using a 1 ml plasma sample volume. The accuracy across the entire range of spiked DEX and DOR concentrations was, in general, within 10% of the spiked value. The precision was generally better than 6% for replicate sample preparations at levels of 50 pg ml-1 or higher and typically better than 12% at levels below 50 pg ml-1. The method was applied for the evaluation of the pharmacokinetic profiles of dextromethorphan and dextrorphan in a human volunteer following peroral administration of a commercially available cough formulation.

A tandem mass spectrometric (ms/ms) method using desorption chemical ionization is described for the quantitation of taxol [1], cephalomannine [2], and baccatin III [3] found in Taxus brevifolia bark and needle extracts. A parent ion scan was used to simultaneously determine the weight percentages of 1-3 in bark and needle samples by the method of standard addition. In an alternative experiment, the concentration of 1 in the same samples was determined by ms/ms using trideuterated 10-acetyltaxol [7a] as an internal standard. High-performance liquid chromatography (hplc) was also used to determine the weight percentages of 1-3 in the same T. brevifolia bark and needle extracts with an external standard. The ms/ms method of quantitation by internal standard is the best overall method of analysis examined. With this method, 1 was quantitated in the T. brevifolia extracts at the low picomole level with a relative standard deviation of 17% or better for all samples analyzed with an analysis time of less than five min per sample. The precision, level of quantitation, and speed of analysis of the three methods of taxane quantitation are compared.

A highly specific and sensitive method is described for determining taxol, cephalomannine, and baccatin III in crude plant extracts. Radical anions of the taxanes are formed by desorption chemical ionization, and a parent tandem mass spectrometric scan is used to recognize these compounds by their characteristic dissociations. The limit of detection of the individual taxanes in typical plant matrices is less than 500 pg when all three species are screened simultaneously. Because of the sensitivity of the method, extraction times can be shortened to 30 min and crude extracts can be examined at the rate of 6/h. Detection of all three taxanes extracted from a single Taxus cuspidata needle in a combined extraction/analysis time of less than 1 h is demonstrated.