Gargi Choudhary, Tania A. Sasaki, and Julian J. Phillips, Thermo Electron Corporation, San Jose, CA
Chromatography and Mass Spectrometry Application Report
Ion trap mass spectrometry is widely recognized for solving analytical problems requiring high sensitivity MSn such as for structural elucidation and identification of unknowns in complex matrices. Furthermore, its full-scan sensitivity is responsible for its utility in screening analysis. Although the popularity of ion trap mass spectrometry is concentrated mostly on qualitative analyses, these mass spectrometers perform well for quantitative analyses as well.
In this report we have used a full-scan MS/MS quantitative assay of antipsychotic drug alprazolam and its analog temazepam spiked in protein precipitated bovine plasma to evaluate the quantitative abilities of a Finnigan LCQ Deca XP Plus ion trap mass spectrometer.
Analytes: Temazepam and alprazolam Internal Standard (ISTD): alprazolam-d5
The proteins from a plasma matrix were precipitated by the addition of an equal volume of 80:20 acetonitrile: methanol containing 1% acetic acid. The plasma sample was then centrifuged for 5 minutes and supernatant decanted. The supernatant was spiked with appropriate levels of analytes and the internal standard.
Standard samples: 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 40, 100, 200, 400, 1000 pg/μL
Internal Standard (ISTD): 10 pg/μL
HPLC system: Surveyor MS pump with Surveyor autosampler
Column: 20 2.1 mm i.d. packed with 5 μm AQUASIL
C18 stationary phase (Thermo Ele ctron).
Injection volume: 5 μL
Flow Rate: 400 μL/min
Mobile phase A: water containing 10 mM ammonium acetate B: acetonitrile containing 0.1 % formic acid
Gradient: 45% B for 3 minutes
Ionization mode: Positive electrospray ionization (ESI)
Capillary temperature: 350C
Spray voltage: 4.5 kV
Sheath gas: 45 units Sweep gas: 10 units
Maximum inject time: 400 ms
Isolation width: 1.0
Collision energy: 40%
Number of segments: 2
Segment 1: Full-scan MS/MS m/z 301.2 → m/z [100-305]
Segment 2: Full-scan MS/MS Scan event 1 m/z 309.2 → m/z [100-311] Scan event 2 m/z 314.2 → m/z [100-320]
The standard samples were analyzed with five replicate injections at each concentration level and a blank (MeOH) injection between each level.
Results and Discussion
Eleven calibration standards over a concentration range of four orders were used to evaluate the quantitative performance of the LCQ Deca XP Plus mass spectrometer. Figure 1 shows the extracted ion chromatograms from full-scan MS/MS of alprazolam (500 fg on-column) and alprazolam-d5 internal standard (50 pg on-column) in protein precipitated bovine plasma. These chromatograms were generated by summing the two most intense product ions observed in the full-scan MS/MS spectra (Figure 2) acquired for the analyte (m/z 274 + 281) and the internal standard (m/z 279 + 286). The LOD was determined to be 500 fg for alprazolam, with a signal-tonoise of 5 to 1.
Figure 3 shows the extracted ion chromatograms from full-scan MS/MS of temazepam and alprazolam spiked in protein-precipitated bovine plasma at 0.2 pg/μL (1 pg oncol umn) and the internal standard alprazolam-d5 at 10 pg/μL (50 pg on-column). Figure 1 shows extracted ion chromatograms of the most intense product ions observed in the full-scan MS/MS spectra for temazepam (m/z 255), alprazolam (m/z 274 + 281), and the internal standard, alprazolam-d5 (m/z 279 + 286). Figure 4 shows the full-scan MS/MS spectra of temazepam, and alprazolam at a concentration level of 1 pg on-column and the internal standard, alprazolam-d5 (50 pg on-column). This concentration level exhibited a signal-to-noise ratio of 7 to 1 for temazepam and 9 to 1 for alprazolam.
The calibration curve for alprazolam over the entire range of the assay is shown in Figure 5, with the linearity over the dynamic range from 500 fg to 5 ng on-column. The correlation coefficient is 0.9991. The precision values for the alprazolam assay are displayed in Table 1. The % CV or RSDs show that the LCQ Deca XP Plus ion trap mass spectrometer is capable of quantitating analytes at low picogram amounts in plasma. Table 2 shows that the limit of detection (LOD) and quantitation (LOQ) for the antipsychotic drug alprazolam are determined to be 500 fg and 1 pg, respectively, whereas that for its analog temazepam are 1 pg and 2.5 pg, respectively.
These measurements demonstrate that the LCQ Deca XP Plus ion trap mass spectrometer can be used to effectively quantify compounds in biological matrices. Benzodiazapines were successfully quantified with linearity over four orders of magnitude for alprazolam. The CV was around 5-7% except near the LOQ, where the CV was 15%. The LOD and LOQ for alprazolam is 500 fg and 1 pg, respectively whereas that for temazepam are 1 pg and 2.5 pg, respectively.