Rapid and Streamlined Screening of Barbiturates

Barbiturates are a class of antidepressants whose abuse and addiction by recreational users has become a widespread problem (www.nlm.nih.gov). In our work we strived to streamline the barbiturate screening process to provide a fast, cost-effective, and reproducible method from start to finish for forensic labs who are involved in high-throughput processing.

Table I: Solid phase extraction: Strata-X-Drug N 100 mg/6 mL (part no. 8B-S129-ECH)

Experimental Conditions

Phenobarbital, butalbital, pentobarbital, amobarbital, and secobarbital were spiked into urine samples at 40, 100, and 125% of cutoff level (300 ng/mL). The spiked urine samples were then subjected to a pretreatment followed by SPE on a 100 mg/6 mL Strata™-X-Drug N tube as specified in Table I. After extraction, the barbiturates were analyzed by LC–MS-MS using a Kinetex® 2.6 μm C18 100 × 2.1 mm core-shell HPLC/UHPLC column with the MS operating in APCI negative mode (Figure 1).

Figure 1: LC–MS-MS analysis of barbiturates.

Results and Discussion

While developing an extraction method for the barbiturate spiked urine samples it was discovered that conditioning the Strata™-X-Drug N SPE sorbent was not necessary as the elimination of this step did not affect recovery. By skipping this step we were able to save both solvent and time which could drastically improve the efficiency of a high throughput lab. The more efficient SPE extraction also provided high recoveries and excellent reproducibility which is noted in Table II.

Table II: Relative recovery, RSD, and linearity of barbiturates

Downstream LC–MS-MS analysis on the Kinetex® core-shell HPLC/UHPLC column also provided significant benefits as we were able to successfully separate pentobarbital and amobarbital, which differ from each other by the placement of a methyl group which has historically made it difficult to resolve the two compounds. It is thought that the separation on the Kinetex column was made possible by the high peak capacity and efficiency of the Kinetex core-shell particle. Core-shell particles contain a 1.9 μm solid core surrounded by a 0.35 μm porous layer of silica. This allows the particle to perform like a sub-2 μm column in terms of resolution, efficiency, and speed without the limiting backpressures that are associated with sub-2 μm particles making it easy to adopt this technology without having to invest in a UHPLC system.

Conclusion

By pairing a streamlined SPE extraction with an efficient LC–MS-MS analysis, it is possible for any forensic lab to improve their barbiturate screening. Strata-X-Drug N SPE sorbent provided both time and solvent savings which are multiplied when screening numerous samples at once, making it faster and more profitable for forensic labs to screen barbiturate samples. After cleanup, a sensitive LC–MS-MS method on Kinetex 2.6 μm C18 core-shell HPLC/UHPLC columns provides separation and resolution of five barbiturates.

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