Evaluation of the Quattro micro for the Quantification of Diazepam in Human Plasma 

The robustness of the Quattro micro Tandem Quadrupole System has been evaluated with diazepam in protein-precipitated human plasma.

In recent years triple quadrupole mass spectrometers have become the instruments of choice for quantitative analysis of drug-related compounds in the pharmaceutical industry. The sensitivity of atmospheric pressure ionization (API) coupled with the specificity and speed of analysis that can be achieved with triple quadrupole mass spectrometers operating in multiple reaction monitoring (MRM) mode make LC-MS/MS the method of choice for quantitative applications.

The Quattro micro is a benchtop tandem quadrupole incorporating a Z SPRAY atmospheric pressure ionization source with a vertically mounted probe. The instrument has been designed as a compact, high performance instrument for optimized high throughput quantification. Experiments have been designed to evaluate the robustness of this interface for the analysis of samples derived from in vivo drug testing.

The robustness of the system has been evaluated with Diazepam in protein-precipitated human plasma.

The proteins from a plasma sample were precipitated by the addition of an equal volume of acetonitrile. The plasma sample was then centrifuged for 5 minutes and the supernatant decanted. Diazepam was spiked into the supernatant at a concentration of 200 pg/μL and 350 repeat injections of the sample were performed. The analytical conditions are described below.

Figure 1 shows the plot of peak area against injection number for the integrated MRM chromatograms from the analysis of diazepam in protein-precipitated plasma. The average response for the detected peak was 54187 with a relative standard deviation of 2.1%. The quantitative analysis of the samples in biological matrices in pre-clinical and clinical studies requires both high throughput and good analytical accuracy and precision. No discernible reduction in response was observed over the analysis period despite all of the injected matrix being passed into the source. The RSD of 2.1% demonstrates excellent reproducibility over the 35-hour analysis period which is easily within the requirements of precision for quantitative bioanalysis.

A calibration series of diazepam in protein-precipitated plasma was prepared over the concentration range of 0.5 pg/μL to 5 ng/μL. These were injected in duplicate and peaks are plotted against concentration. A plot of peak area against concentration shows good linearity over the range 0.5 pg/μL to 5 ng/μL. The calibration line was plotted using a linear fit with 1/x weighting and resuted in a coefficient of determination of 0.9984 (Fig. 2.)

The Quattro micro tandem quadrupole system has been developed for quantitative LC-MS/MS. The instrument has been evaluated by analyzing a diazepam in protein-precipitated human plasma over a period of 35 hours (350 injections) in order to show its robustness and tolerance to protein-precipitated plasma and the linear increase in response with concentration. 

The results showed that with over 350 injections of the Diazepam the sensitivity was maintained with a relative standard deviation of 2.1%. The calibration line resulted in a linear plot over the range 0.5 pg/μL to 5 ng/μL (Figure 2) with a correlation coefficient of 0.9984 demonstrating that the Quattro micro shows good linearity over 4 orders of magnitude.