Flualprazolam was purchased from Cayman (Michigan, USA); 2-chlorodiazepam and diazepam-d5 (used as internal standard, IS) were from Sigma (MO, USA), all of HPLC-pure grade. Acetonitrile and methanol, products of Fisher Scientific (Fairlawn, NJ, USA). Formic acid, from Sigma-Aldrich (USA). ISOLUTE SLE+ 1 mL supported liquid extraction column, from Biotage (Charlotte, North Carolina, USA). Deionized water was generated with an Elix water purification system (Millipore, Molscheim, France). All glass vials and vial inserts were silanized and the screw caps were PTFE-lined, with all of them purchased from VWR International (Mississauga, Ontario, Canada).

Analyses were performed on an Agilent 1290 HPLC system with the SCIEX QTRAP 6500 MS/MS as the detector. Chromatographic data were recorded and analyzed by Analyst^® software.

Separations were carried out on a Kinetex C18 column (3.0 mm× 100 mm, 2.6 μ m; PHENOMENEX, USA) that was maintained at the temperature of 35 ° C and run under a constant flow rate of 0.3 mL/min. The mobile phase was composed of 5 mmol/L ammonium formate in purified water (A) and acetonitrile (B), with both of them containing 0.1% formic acid. The selected gradient program was shown in Table 1. The sample-injecting volume was 1 μ L.

Table 1

Table 1

Table 1 Gradient program for HPLC Time/min Flow/(mL/min) A/% B/% 0.0 0.3 5 95 1.0 0.3 5 95 10.0 0.3 95 5 13.0 0.3 95 5 13.5 0.3 5 95 15.0 0.3 5 95

Table 1 Gradient program for HPLC

The quantitative analysis of flualprazolam and 2-chlorodiazepam was performed in MRM mode. Positive ionization mode (ESI⁺) was used for all analytes. The MS parameters were: curtain gas, 35 psi; ionspray voltage,
5 500 V; temperature, 550 ° C; ion source gas1, 55 psi; ion source gas2, 55 psi; collision gas, 10 psi. Each analyte was identified via two characteristic MRM transitions as shown in Table 2.

Table 2

Table 2

Table 2 Mass spectrometric parameters for analytes and internal standard (IS) Analyte PI/(m/z) DI/(m/z) CXP/V CE/V DP/V flualprazolam 327.0 299.1 10 35 130 327.0 223.0* 10 46 130 2-chlorodiazepam 319.1 227.1 10 40 40 319.1 154.3* 10 40 40 diazepam-d5 (IS) 290.1 198.1 10 43 60 290.1 227.2 10 38 60 PI: parent ion; DI: daughter ion; CXP: collision cell-exit potential; CE: collision energy; DP: declustering potential; * : quantitative ion

Table 2 Mass spectrometric parameters for analytes and internal standard (IS)

For blank samples, human venous whole bloods were obtained from a Fujian provincial hospital (Fuzhou, Fujian, China). These samples were screened before used as the negative controls, the blanks with internal standard, the spiked calibrators and the quality control samples. Authentic forensic samples of whole blood were preserved with sodium fluoride (Merck, Darmstadt, Germany) and potassium oxalate (BD, Plymouth, UK).

2.5.1 SLE

ISOLUTE SLE+ 1mL columns were used for blood sample preparation. 500 μ L sample of spiked blood (added of 1 ng/mL diazepam-d5, the IS) was transferred into an SLE+ column. A minimum positive pressure from an in-house system (0.1 bar) was applied to facilitate the sample to absorb into the cartridge in less than 10 s. After the analytes were allowed to equilibrate with the sorbent for a minimum of 5 min, the compounds were eluted with 1mL × 2 of ethyl acetate. The eluate was evaporated to dry under nitrogen at 45° C. The dried samples and blank samples were reconstituted with 400 μ L of acetonitrile/water (25/75, v/v) and standard solution of analytes in acetonitrile/water (25/75, v/v), respectively, and vortexed to mix for 2 min. The resulting solution was directly subject to LC-MS/MS analysis.

2.5.2 LLE

500 μ L sample of spiked blood (containing 1 ng/mL diazepam-d5, IS) was prepared through a liquid-liquid extraction with ethyl acetate/heptane mixture (4∶ 1, v/v) after the addition of 0.1 mol/L borate buffer (pH 9) and followed to be shaken for 5 min. Subjected to centrifugation at 3 000 r/min for 10 min, the extracted solution of spiked blood sample had its upper layer transferred into another test tube. With the above extraction step being repeated for three times with the 500 μ L spiked blood sample, the combined extract was evaporated to dry at 45 ° C under nitrogen gas. The dried sample was reconstituted with 400 μ L of acetonitrile/water (25/75, v/v) and subjected to LC-MS/MS analysis.

2.5.3 SPE

The blood samples were diluted with 3 mL of 0.1 mol/L phosphate buffer (pH 7.2), vortexed for 15 s, and then sonicated for 15 min. Resulted from centrifugation at 3 000 r/min for 5 min, the supernatant was loaded onto the HLB SPE cartridge preconditioned with 2 mL of at-first methanol and then distilled water. After loaded, the cartridge was washed with 2 mL of 5% methanol and the analytes were subsequently eluted with 3 mL of 20% methanol in water. Subsequently, the eluates were evaporated to dry under nitrogen at 45 ° C. The dried sample was reconstituted with 400 μ L of acetonitrile/water (25/75, v/v) and subjected to LC-MS/MS analysis.

The specificity and selectivity were tested with separate samples from two different human blood samples spiked. Fig.2 showed the total ion chromatograms (TIC) of flualprazolam, 2-chlorodiazepam and diazepam-d5 through LC-MS/MS, with them being eluted out at 7.70, 9.02 and 8.87 min, respectively. No peak was detected in the blank blood sample, and there was no interfering peaks derived from endogenous substances at the elution time of the analytes.

Fig.2

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	Fig.2 LC/MS total ion chromatograms (TIC) of an extracted blood sample spiked with flualprazolam, 2-chlorodiazepam and diazepam-d5

The extraction methods of LLE, SPE and SLE were compared. As shown in Fig.3, the extraction efficiencies for flualprazolam and 2-chlorodiazepam were improved most significantly with SLE. Therefore, SLE was selected to treat the sample.

Fig.3

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	Fig.3 Comparison of sample cleanup methods

The results of the matrix effect, recovery and processing efficiency of flualprazolam and 2-chlorodiazepam in human whole blood were summarized in Table 3. The mean matrix effect was 77.5% for flualprazolam and 72.5% for 2-chlorodiazepam. The mean recovery was 95% for flualprazolam and 79% for 2-chlorodiazepam. The mean processing efficiencies of flualprazolam and 2-chlorodiazepam were 73% and 72%, respectively.

Table 3

Table 3

Table 3 The matrix effect, recovery and processing efficiency of flualprazolam and 2-chlorodiazepam Analytes Concentration /(ng/mL) Matrix effect /% Recovery /% Process efficiency /% flualprazolam 20 77.1 95 73 200 79.3 101 71 400 76.2 89 76 mean 77.5 95 73 2-chloro- diazepam 20 75.8 77 71 200 70.8 82 75 400 71 79 70 mean 72.5 79 72

Table 3 The matrix effect, recovery and processing efficiency of flualprazolam and 2-chlorodiazepam

The calibration curves showed good linearity over the concentrations ranging among 5.0-500 ng/mL, with correlation coefficient (R²) greater than 0.99. The mean slopes and intercept values of calibration curves for the flualprazolam and 2-chlorodiazepam were given in Table 4. The LOD values of flualprazolam and 2-chlorodiazepam were 0.05 ng/mL and 0.1 ng/mL, respectively. The low limit of quantification was 5 ng/mL for the two analytes, thus being defined as the lowest concentration in the calibration curve with acceptable precision and accuracy (± 20% bias).

Table 4

Table 4

Table 4 Linearity and LODs for flualprazolam and 2-chlorodiazepam Analytes Concentration /(ng/mL) R2 Slope Y-intercept LOD /(ng/mL) flualprazolam 5-500 0.9995 0.003 -0.037 0.05 2-chloro- diazepam 5-500 0.9991 0.002 0.015 0.1

Table 4 Linearity and LODs for flualprazolam and 2-chlorodiazepam

The intra- and inter-day precision and accuracy were summarized in Table 5. Precision (RSD) was shown as the coefficient of variation (CV%) of the concentrations determined by replicates of the QC samples. The supported liquid-liquid extraction method here-developed showed good precision and accuracy. From both the intra- and inter-day analyses, the accuracy ranged from 86.0% to 109.0% at low, medium or high concentrations, with the precision (RSD) values of CV% less than 6.2%.

Table 5

Table 5

Table 5 Precision and accuracy for flualprazolam and 2-diclazepam in human whole blood spiked Analytes Concentration /(ng/mL) Intra-day (n = 5) Inter-day (five different days) Measured conc. (mean ± SD, ng/mL) RSD/% Accuracy /% Measured conc. (mean ± SD, ng/mL) RSD /% Accuracy /% flualprazolam 20 19.0 ± 0.7 3.7 95.0 19.1 ± 0.7 3.5 95.4 200 204.1± 4.8 2.4 102.1 217.4± 10.1 4.6 109.0 400 370.6± 7.5 2.0 92.7 364.7± 9.6 2.6 91.2 2-chlorodiazepam 20 18.6 ± 1.0 5.4 93.0 17.2 ± 0.7 4.1 86.0 200 201.2± 1.2 0.6 100.6 211.0± 13.1 6.2 106.0 400 381.8± 5.4 1.4 95.5 378.6± 17.5 4.6 94.7

Table 5 Precision and accuracy for flualprazolam and 2-diclazepam in human whole blood spiked

The established method was applied into several real forensic cases. Bloods from two sexually assaulted victims were collected for toxicological analysis. Flualprazolam and 2-chlorodiazepam were successfully detected in all samples, with their concentrations shown in Table 6.

Table 6

Table 6

Table 6 Details of forensic cases and detection results from the established method Case No. Sex/age (years) Detected compound Concentration /(ng/mL) 1 f/20 Flualprazolam 4400 2 f/23 2-chlorodiazepam 1020

Table 6 Details of forensic cases and detection results from the established method