Standards of lorazepam, clonazepam, bromazepam, flunitrazepam, alprazolam, temazepam, chlordiazepam, oxazepam, diazepam, 7-aminoflunitrazepam, nitrazepam and diazepam-d5 (internal standard, IS) were purchased from Cerilliant (Round Rock, TX, US). Individual drug standards were prepared at 1 mg/mL in pure methanol, being stored at -20 ° C. All reagents were of HPLC grade. Acetonitrile and methanol were products from Fisher Scientific (Fairlawn, NJ, US). Formic acid was obtained from Spectrum Chemical MFG Corp. (Gardena, CA, US). Deionized water was generated with the Millipore Elix water purification systems (Millipore Corp., Molscheim, France).

Methanolic mixed standard solutions (1.0 μ g/mL) were prepared from each drug standards (1.0 mg/mL). The working standard solutions were prepared from the mixed standard solution through dilution to the appropriate concentrations as required. The working solutions were stored at -20 ° C.

Blank hair samples were obtained from healthy volunteers with no history of drug abuse. The donors have signed the informed consent. Hair samples were stored in a clean envelope at room temperature. All the procedure regarding with the sample collection was approved by the Ethics Committee on Human Research of the Faculty of Health Science.

Prior to analysis, the hair samples were firstly washed with shampoo and secondly rinsed with deionized water then acetone and finally air-dried overnight. The washing solutions were analyzed via the novel microfluidic LC-MS/MS methods to secure no presence of contamination. Dried hair sample was cut into 1 mm pieces and ground with a ball-grinder (Retsch, Haan, Germany). 20 mg of ground hair sample was weighed in a glass tube, with addition of 100 μ L of mixed standard solution and 2 mL of boric acid buffer (pH 9.5). The mixture was ultrasonicated for 30 minutes before addition of 2 mL of extractant (dichloromethane/ether/hexane=30/50/20), being undergone with vertex for 10 minute. The extract was centrifuged at 10 000 r/min for 5 minutes. The supernatant was transferred to a clean Eppendorf tube and was evaporated under N₂ stream until almost dry. The residue was reconstituted by 50 μ L of methanol/water (1꞉9), with 1.0 μ L for the analysis by either the novel microfluidic LC-MS/MS or the traditional LC-MS/MS system.

The analysis of 11 target drugs was performed with Waters ACQUITY UPLC M-class combined into the Xevo TQ-S mass spectrometer that was fitted with an IonKey source. The analytes were separated on an iKey peptide BEH C18 column (150 μ m× 50 mm, 1.7 μ m; Waters, US), which was held at 45 ° C. Mobile phase A was water with 0.1% formic acid. Mobile phase B was consisted of methanol (containing 0.1% formic acid) and acetonitrile (3꞉1). The mobile phase was changing from the initial of 10% B to the final 95% B in 6 min and kept for 2 min, followed by a descending linear gradient to 10% B in 0.5 minutes and held constant for 4.5 minutes. The flow rate was 3 μ L/min. The UPLC M-class system was connected to Xevo TQ-S MS equipped with an IonKey source in positive mode. The MS parameters were listed as: capillary voltage 3.8 kV, the source temperature 150 ° C and the cone gas flow 50 L/h. 1 μ L of the hair extract was subjected into the novel microfluidic LC-MS/MS system and the chromatographic data were recorded by Masslynx^TM software (Version 4.1).

The analysis was performed by Waters ACQUITY UPLC^TM coupled with Xevo TQ-S mass spectrometer (ESI source). The 11 target drugs were separated through a BEH C18 column (2.1μ m× 50 mm, 1.7 μ m; Waters, USA), which was maintained at 45 ° C. Mobile phase A and B were the same as those mentioned in section 2.5. The gradient was slightly adjusted. The initial mobile phase composition (2% B) was kept for 0.5 minutes and increased to the 95% B in 5.5 minutes with another 2 minutes kept, followed by a descending gradient to 2% B in 7 minutes and held constant for 2 minutes. The flow rate was 0.5 mL/min. The UPLC system was connected to the Xevo TQ-S MS equipped with ESI source in positive mode. The MS parameters were the following: capillary voltage 3.0 kV, the source temperature 150 ° C , the desolvation temperature 500 ° C and the desolvation gas flow 800 L/h. 1 μ L of the hair extract was undergone into the traditional LC-MS/MS system and the chromatographic data were recorded by Masslynx^TM software (Version 4.1). The two transitions per analyte and respective setting for LC-MS/MS system were given in Table 1.

Table 1

Table 1

Table 1 MRM transitions and respective settings for each analyte Analyte Precursor ion Product ion Cone voltage /V Collision energy /V Analyte Precursor ion Product ion Cone voltage /V Collision energy /V Lorazepam 321.0 275.1/303.1 52 18/14 Chlordiazepam 300.1 227.0/283.0 62 36/16 Clonazepam 316.1 270.1/214.1 44 24/36 Oxazepam 287.1 241.2/269.2 20 18/14 Bromazepam 316.1 182.2/209.1 26 28/24 Diazepam 285.1 193.2/154.2 56 26/34 Flunitrazepam 314.1 268.2/239.2 2 24/34 7-aminoflunitrazepam 284.2 135.0/226.9 24 26/22 Alprazolam 309.2 281.2/205.1 38 38/26 Nitrazepam 282.1 236.2/186.1 52 24/40 Temazepam 301.1 255.1/193.1 28 34/38

Table 1 MRM transitions and respective settings for each analyte

There are several factors affecting the extraction efficiency, e.g., ultrasonic time, extraction solvent and extraction duration. The influence of these factors was studied and optimized. A KQ-250E ultrasonic apparatus (Kunshan, China) was used for isolation of the 11 benzodiazepines from hair sample. 100 μ L of mixed standard solution was spiked into 20 mg of ground hair. Then, 2 mL of boric acid buffer was added as reported from literature ^[19] for reference. The ultrasonic process was carried out at 50 W for 10, 20, 30, 40 and 50 minutes, respectively. It was showed that extraction efficiency achieved best at 30 minutes (shown in Fig.1).

Fig.1

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	Fig.1 The effect of ultrasonic time on extraction efficiency

The liquid-liquid extraction process was another important factor for the extraction efficiency. Several solvents such as ethyl ester, dichloromethane, ether and hexane were examined. According to the results, a mixture of dichloromethane/ether/hexane (30/50/20) was found to get the highest extraction efficiency and thus selected as the extractant. The influence of liquid-liquid extraction time was also optimized in the range of 5~20 min, demonstrating that the extraction efficiency remained constant after 10 min. Therefore, 10 min was chosen for the following work (shown in Fig.2).

Fig.2

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	Fig.2 The effect of extraction solvent/time on extraction efficiency

According to FDA guidelines, both the novel microfluidic LC-MS/MS and the traditional LC-MS/MS were validated on the items of their selectivity, specificity, linearity, limit of quantification (LOQ), limit of detection (LOD), precision, accuracy, extraction efficiency and matrix effect. Selectivity and specificity were evaluated with analysis of six different sources of blank hair samples and spiked hair samples at the concentration of 10 pg/mg through the novel microfluidic LC-MS/MS method and the traditional LC-MS/MS choice, respectively. Mean area measurements for the selected analyte ions were generated and analyzed to ensure that the matrices were free from interfering compounds. Calibration curves were prepared by the spiking of appropriate mixed standard solution into 20 mg of blank hair at concentrations of 0.005, 0.012 5, 0.025, 0.05, 0.125, 0.25, 0.5, 1.0, 2.5, 5.0 and 10 pg/mg for each analyte. Calibration was performed through linear regression of peak-area ratio of the targeted drugs and IS versus the respective standard concentration. LOQs corresponding to the lowest concentrations were used for the calibration curves under a signal-to-noise ratio of at least 10. The resulting linearity ranges, correlation coefficients, LOQs and LODs of each analyte were reported in Supplementary Table S1. A representative chromatogram of LODs for each compound was showed in Fig.3.

Fig.3

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	Fig.3 Quantitative ion chromatograms of extracted hair samples harboring the 11 drugs at LOQ

The precision and accuracy were evaluated at three concentration levels: lowest, middle and highest points of the calibration curve from standards. The intra- and inter-day precisions were evaluated via analysis of five replicates of each level for three consecutive days. The accuracy was the percent of calculated concentration value at a specific level from the respective nominal concentration. Data of precision and accuracy were listed in Supplementary Table S2.

Ion suppression was estimated for each analyte through comparison of the peak area obtained from hair samples spiked before extraction, against that of a methanolic standard at the responding concentration. The extraction efficiency was determined for each analyte through comparison of the peak area obtained from hair samples spiked before extraction, against that obtained from hair samples spiked after extraction. The data were showed in Supplementary Table S3.

Both the novel microfludic LC-MS/MS method and the traditional LC-MS/MS have a good linear relationship for each analyte, with their correlation coefficients all above 0.99. For the novel microfluidic LC-MS/MS, it was showed of a wide linear range. All of the analytes displayed 2~10 folds higher of LODs from the novel microfluidic LC-MS/MS method than the traditional LC-MS/MS one. The intra- and inter-day precisions via the two LC-MS/MS methods were comparable and within the required limits of 15% RSD. Accuracy ranged from 88.4% to 113.6% for the novel microfluidic LC-MS/MS method and from 93.6% to 110.9% for the traditional LC-MS/MS, respectively. Supplementary Table S4 showed that the extraction efficiency and matrix effect were also satisfactory.

Early in 1997, V. Cirimele et al^[2] presented a GC-MS method for the detection of human hair of forensically relevant benzodiazepines. The LODs only reached 1~20 pg/mg. A. Salomone et al^[5] developed and validated a specific LC-MS/MS protocol for determining benzodiazepines at low concentration in hair specimens. For diazepam and bromazepam, the sensitivity was improved significantly. C. Montesano et al^[16] established a UPLC-MS/MS assay for analysis of 96 drugs including benzodiazepines and the LODs ranging from 1 to 20 pg/mg. Recently, A. Woź niakiewicz et al^[22] developed a quick CE-MS/MS method for simultaneous determination of benzodiazepines in hair samples. The LODs for benzodiazepines were within 6~23 pg/mg. Obviously, the sensitivity of reported methods could not fit for our purpose about trace amount of benzodiazepines detection. Both clear and intuitive comparison data were summarized in Supplementary Table S5. An effective increase was observed in sensitivity with the developed microfluidic LC-MS/MS method as compared to published literatures. With the ultrasensitive microfluidic LC-MS/MS method, more information about long-term assumption would be provided through hair analysis.

The sensitivity improvement of the microfluidic LC-MS/MS method is attributed to the novel ionKey source. Firstly, an electrospray plume generated from conventional LC flow rates can be quite broad and divergent. As the solvent is reduced of its flow rate, the electrospray plume decreases in size and becomes more convergent. This allows the inlet of the mass spectrometer to become more efficient and capture a greater percentage of the plume, resulting in the increase of ion signal. Secondly, smaller charged droplets are produced by ionKey source so that ions are much more preferential to gather on the surface of charged droplets and are therefore further more prone to be gasified. This improves the ionization efficiency. Thirdly, the decreasing droplet size causes fewer interfering charged ions when ionization in each droplet and thus ion suppression is reduced. Based on the three points mentioned above, a dramatic sensitivity improvement has been successfully achieved by the microfluidic LC-MS/MS method.

A hair sample was collected from a 45-year-old woman who had taken 1mg of Flunitrazepam every night to treat insomnia for 2 weeks. She has no other medical history. The hair sample was cut close to the scalp from the posterior vertex area of the woman’ s head, and prepared according to the above-mentioned protocol in section 2.4. Subsequently, the extract was submitted for analysis. Flunitrazepam and its metabolite 7-aminoflunitrazepam were detected, with their concentrations being respective of 1.5 and 4.2 pg/mg, such low detection limits that the traditional methods are usually unable to reach.