The determination of nicotinic acid in plasma by mixed-mode liquid chromatography-tandem mass spectrometry following ion exchange solid phase extraction.

An assay for nicotinic acid in plasma samples has been developed using ion exchange solid phase extraction in 96-well format followed by mixed-mode ion exchange/reversed-phase liquid chromatography with positive ion tandem mass spectrometry detection. The assay avoids the need for time-consuming derivatisation procedures or involatile ion-pair chromatography reagents. The assay is linear over the wide range 0.05-20 microg/mL, based on a 100 microL sample (correlation coefficient>0.99). The assay is accurate and precise (bias and coefficient of variation<18%) over this calibration range.

Quantitative analysis of pharmaceuticals in biological fluids using high-performance liquid chromatography coupled to mass spectrometry: a review.

1. The development of bio-analysis of drug molecules over the last 10 years is reviewed, focusing on advances in sample preparation, liquid chromatography and detection. 2. Developments have led to improvements in detection sensitivity, enhancements in specificity and increased capacity. 3. Emerging technologies such as monolithic column chromatography and miniaturized chip-based systems are discussed.

Determination of 4-hydroxytamoxifen in mouse plasma in the pg/mL range by gradient capillary liquid chromatography/tandem mass spectrometry.

Capillary high-performance liquid chromatography (HPLC; 300 microm i.d.) coupled to tandem mass spectrometry has been used to determine the concentration of 4-hydroxytamoxifen in mouse plasma in the pg/mL range following the administration of Tamoxifen. A limit of quantification (LOQ) of 100 pg/mL was achieved using only 25 microL of plasma. The on-column sensitivity was determined to be 100 fg. The column performance was determined isocratically before and after the assay and showed only a 15% reduction in performance after 70 injections of plasma extract. No significant peak band broadening was observed due to the mass spectrometer interface using a standard TurboIonspray source.

Parallel ultra-high flow rate liquid chromatography with mass spectrometric detection using a multiplex electrospray source for direct, sensitive determination of pharmaceuticals in plasma at extremely high throughput.

Recent years have seen increasing usage of large particle size stationary phases and ultra-high flow rate liquid chromatography/mass spectrometry (LC/MS) for rapid determination of pharmaceuticals in plasma without prior sample preparation. This lack of sample preparation prior to analysis, together with the extremely high throughput of the chromatography, makes the technique extremely attractive to the bioanalyst. Further, the introduction of multiple sprayer interfaces to mass spectrometers provides the potential for even higher throughput. In this paper, we present parallel ultra-high flow rate liquid chromatography using four columns in parallel and a four-way multiple sprayer interface to the mass spectrometer. We have applied this on both the narrow-bore and capillary scale. This technique enables the quantification of drugs from four plasma samples simultaneously, at nanogram per millilitre concentrations, from small aliquots of plasma without sample preparation and with throughputs of up to 120 samples per hour.

Ultra-high flow rate capillary liquid chromatography with mass spectrometric detection for the direct analysis of pharmaceuticals in plasma at sub-nanogram per millilitre concentrations.

Ultra-high flow rate liquid chromatography on large particle size stationary phases coupled with mass spectrometric detection (particularly tandem mass spectrometry, MS/MS) is gaining increasing usage for the direct determination of pharmaceuticals in biological fluids. The lack of sample preparation required prior to chromatographic and MS/MS analysis, together with the extremely high throughput of the chromatography, make the technique extremely attractive to the modern pharmaceutical bioanalyst. However, this lack of sample preparation also means that there is no potential for concentration of the sample and, as a consequence, the sensitivity of the technique has been limited. Liquid chromatography on the capillary scale offers sensitivity benefits compared with conventional liquid chromatography as the volume in which the analyte peaks are eluted is greatly reduced. In this paper, we present the use of ultra-high flow rate liquid chromatography on the capillary scale. This enables the quantification of drugs in plasma at sub-nanogram per millilitre concentrations from a very small (2.5 micromgL) aliquot of plasma without sample preparation. We also compare the resolution obtained by ultra-high flow rate liquid chromatography with that achieved on short columns packed with conventional size packing materials operated in an isocratic manner.

Use of generic fast gradient liquid chromatography-tandem mass spectroscopy in quantitative bioanalysis.

Short narrow analytical HPLC columns have been used successfully with high linear flow-rates and combined with mass spectrometric detection to produce a generic approach to quantitative bioanalysis. The approach has been used to validate several assays in the low ng/ml region and an example is given in this paper. When combined with a simple solid-phase extraction process the need for complicated, time consuming method development has been removed for the majority of pharmaceutical compounds. The approach takes advantage of not only the extra selectivity of the MS-MS detector but the excellent resolution and peak shape produced by gradient elution.

Optimisation and routine use of generic ultra-high flow-rate liquid chromatography with mass spectrometric detection for the direct on-line analysis of pharmaceuticals in plasma.

The use of ultra-high flow-rate chromatography coupled to mass spectrometry offers great potential for the rapid, on-line analysis of pharmaceutical compounds in plasma as it permits high throughput direct analysis of plasma samples without any time-consuming sample preparation such as solid-phase extraction. The coupling of mass spectrometry with high-performance liquid chromatography often results in enhanced selectivity and sensitivity compared to, for example, ultraviolet absorbance detection. This can remove the need for complete resolution of the analyte from endogenous materials in the matrix. The use of large particle size stationary phases, and therefore, the ability to use large porosity column end frits, coupled with the added selectivity and sensitivity of the mass spectrometer allows an on-line analysis approach to be used for the direct analysis of pharmaceuticals in biological matrices with extremely high throughput. This paper presents an overview of the manner in which we have optimised this technique for the analysis of plasma samples, in terms of gradient profile, system configuration and optimal injection volume for maximum throughput and robustness. The nature of the mobile phase flow is also discussed.

The use of turbulent flow chromatography/mass spectrometry for the rapid, direct analysis of a novel pharmaceutical compound in plasma.

The use of turbulent flow chromatography coupled to mass spectrometry (turbulent flow LC/MS) shows great potential for the rapid, direct analysis of pharmaceutical compounds in plasma and serum. The use of turbulent flow LC/MS has removed the need for any time-consuming sample preparation such as solid phase extraction, and allowed a total sample analysis time of approximately 2.5 min to be achieved. The coupling of a mass spectrometer with HPLC often not only results in greater sensitivity, but also the added specificity of the mass spectrometer reduces the need for complete resolution of the analyte from endogenous material in the matrix. This allows an on-line analysis approach to be used for the analysis of pharmaceuticals in biological matrices. Turbulent flow chromatography is achieved by the use of high flow rates and large particle size stationary phases. When coupled with mass spectrometric detection, the technique allows the direct analysis of plasma or serum samples with very rapid chromatography and, therefore, extremely high throughout. This work demonstrates the suitability of this technique for the validated analysis of biological samples for a novel isoquinoline pharmaceutical and offers some ideas on the future continued development, optimization and application of turbulent flow liquid chromatography.

The minimal metabolism of inhaled 1,1,1,2-tetrafluoroethane to trifluoroacetic acid in man as determined by high sensitivity 19F nuclear magnetic resonance spectroscopy of urine samples.

In this work, oxidative metabolism of the new propellant, 1,1,1,2-tetrafluoroethane to trifluoroacetic acid in man is shown to be minimal. Alternative propellants and refrigerants are under development to replace the currently used chlorofluorocarbons which lead to stratospheric ozone depletion. One potentially useful replacement is the hydrofluorocarbon, 1,1,1,2-tetrafluoroethane (HFA-134a). Before it can be used, however, particularly as a propellant in an aerosol pharmaceutical formulation whereby the compound is in effect dosed to people, it is important that the safety of this compound is established. As a part of this safety evaluation it is necessary to understand the metabolism of HFA-134a. In this work the production of the potential oxidative metabolite of HFA-134a, trifluoroacetic acid (TFA) has been studied in human urine following inhalation dosing with HFA-134a. The concentrations of TFA in urine have been measured using a highly sensitive 19F nuclear magnetic resonance procedure with a limit of detection of 10 ng ml-1 based on an acquisition time of only 2.25 h per sample. TFA is the only fluorinated species observed in the urine samples and only at very low levels, indicating that the oxidative route of metabolism can occur in vivo in man, but this metabolism is minimal in terms of percentage of administered dose.

Demonstration of the compatibility of narrow-bore packed column high-performance liquid chromatography with conventional detection systems.

A number of commercially available high-performance liquid chromatography detectors fitted with conventionally sized flow cells (4.5 to 14 microliters) have been evaluated for use for narrow-bore (1-3 mm i.d.) packed columns. Contrary to the popular misconception, small flow cell volume (and consequently short path length) does not appear to be a prerequisite for successful use of narrow-bore columns. The 1 mm i.d. columns when used with certain detectors gave up to a 17-fold increase in sensitivity, compared with conventional 4.6 mm i.d. columns. This work, which was carried out using conventional injection volumes (50 microliters), shows that narrow-bore HPLC columns can be used in most laboratories to significant advantage without costly conversion of existing equipment.