Urine Barbiturate Analysis Using an Improved SPE Protocol and LC/MS/MS; Achieving Chromatographic Resolution of Isobaric Amobarbital & Pentobarbital Michael Rummel, Matthew Trass, Seyed Sadjadi, Jeff Layne, Sky Countryman, and Erica Pike Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA P89240811_L
Introduction Barbiturates are a notoriously difficult class of compounds to analyze in drugs of abuse testing panels. These neutral and fairlypolar compounds are difficult to retain on conventional Solid Phase Extraction (SPE) sorbents, limiting the wash strength and subsequent matrix cleanup. Additionally, two of the commonly screened barbiturates, pentobarbital and amobarbital, share the same mass transitions meaning full chromatographic resolution is necessary for LC/MS/MS analysis 1, 2. Achieving full resolution is a difficult task as the two barbiturates differ only in the position of a methyl group. Therefore, the goal of this work was to develop a streamlined SPE extraction for barbiturates in urine that removed potential matrix contaminants and a LC/MS/MS method that resolved pentobarbital, amobarbital, butalbital, secobarbital, and phenobarbital. Much emphasis was placed on the sensitivity and reproducibility of the method over a linear range from 40-125 % of the cutoff concentration set at 300 ng/ml.
Figure 1. Barbiturate Structures and Chemical Properties H 2 C N H HN H 3 C H N N H H N N H H 3 C N H H N N H HN Amobarbital logp: 1.88 Mass: 226.2722 Pentobarbital logp: 1.88 Mass: 226.2722 Butalbital logp: 1.56 Mass: 224.2563 Secobarbital logp: 2.01 Mass: 238.2829 Phenobarbital logp: 1.38 Mass: 232.2353 Note: Amobarbital and Pentobarbital are structural isomers.
Experimental Conditions Table 1. Extraction Method and HPLC Conditions Pre-treatment: To 2 ml of urine, add 2 ml of 100 mm Sodium acetate buffer (ph 5.0). ph should be ~4.0-6.0. Cartridge: Strata -X-Drug N, 100 mg/6 ml Part No.: 8B-S129-ECH Condition: Not Required Equilibrate: Not Required Load: Pre-treated urine samples Wash 1: 2 ml 0.1 N Hydrochloric acid (HCl) Wash 2: 2x 2 ml Methanol / 0.1 N HCl (30:70) Dry: 10 minutes at 10 in. of Hg Elute: 2 ml Ethyl acetate/isopropanol (85:15) Dry down: To dryness at 50 C Reconstitute: 1 ml of 10 % Acetonitrile Column: Kinetex 2.6 µm C18 Dimensions: 100 x 2.1 mm Part No.: 00D-4462-AN Mobile Phase: A: 5 mm Ammonium acetate buffer B: Acetonitrile Gradient: Time (min) B (%) 0 10 10 45 10.01 90 12 90 12.01 10 16 10 Flow Rate: 400 µl/min Detection: Negative mode (ESI) Temperature: Ambient
Table 2. LC/MS/MS Parameters (API 5000 ) CUR: 40.00 GS1: 60.00 GS2: 50.00 TEM: 550.00 ihe: N IS: -2500.00 CAD: 4.00 EP -10.00 CXP -13.00 Compound Q1 Mass (Da) Q3 Mass (Da) Dwell (msec) Amobarbital-1 225.00 182.10 100.00-66.00-19.00 Amobarbital-2 225.00 42.10 100.00-50.00-46.00 Butalbital-1 223.10 180.00 100.00-40.00-15.00 Butalbital-2 223.10 42.10 100.00-40.00-40.00 Pentobarbital-1 225.40 182.20 100.00-66.00-20.00 Pentobarbital-2 225.40 42.10 100.00-50.00-46.00 Phenobarbital-1 231.10 85.00 100.00-33.00-17.00 Phenobarbital-2 231.10 42.10 100.00-33.00-40.00 Secobarbital-1 237.00 194.20 100.00-44.00-18.00 Secobarbital-2 237.00 42.10 100.00-59.00-49.00 Butalbital-D5 228.00 41.60 100.00-40.00-15.00 Phenobarbital-D5 235.90 41.80 100.00-33.00-17.00 DP CE
Table 3. Comparison of Solid Phase Extraction Procedures Used in this Study Versus Traditional C8+SCX Sorbent Methods Step Strata-X-Drug N 100 mg/6 ml Part No. 8B-S129-ECH Mixed-Mode Silica-Based SPE Sorbent 200 mg/10 ml Strata-X-Drug N Solvent Savings Condition 0 ml 3 ml 3 ml Equilibrate 1 0 ml 3 ml 3 ml Equilibrate 2 0 ml 1 ml 1 ml Load Pre-treated urine sample Pre-treated urine sample -- Wash 1 2 ml 3 ml 1 ml Wash 2 4 ml 1 ml -3 ml Dry -- -- -- Wash 3 0 ml 2 ml 2 ml Elute 2 ml 3 ml 1 ml TTAL SLVENT SAVINGS 8 ml per sample
Results and Discussion Barbiturate analysis has always been one of the more challenging drug classes to analyze for the toxicologist. With amobarbital and pentobarbital being structural isomers (Figure 1), separation using liquid chromatography requires a combination of excellent selectivity and high efficiency. Furthermore, extracting this drug class on traditionally used C8+SCX SPE sorbents requires very low wash strengths to prevent analyte loss and poor recovery. This results in an inadequate matrix cleanup. Development and optimization of the SPE procedure was performed using 100 mg/6 ml Strata-X-Drug N cartridges. 2 ml urine samples were prepped at three concentrations, (120 ng/ml, 300 ng/ml, and 375 ng/ml (n=3) at each concentration) to establish linearity of the method. Three additional samples were prepared at 300 ng/ml and measured against an extracted matrix matched standard to establish absolute recovery and reproducibility of the extraction. Strata-X-Drug N requires no conditioning allowing for direct sample loading and the elimination of three steps as compared to a traditional procedure. Following the sample load, 2 washes of 0.1N Hydrochloric acid (HCl) and 30 % Methanol (MeH) in 0.1N HCl, a dry step, and an elution of (85:15) (v:v) Ethyl Acetate:Isopropanol were chosen. The use of a 30 % MeH wash is much higher in strength than conventional procedures and resulted in excellent matrix cleanup measured by a clean baseline and a visually clean sample, resulting in time and solvent saving 3 (Table 3). Post extraction samples were analyzed via LC/MS/MS using an Agilent 1200 LC fitted with a Kinetex 2.6 µm C18 100 x 2.1 mm core-shell HPLC/UHPLC column. The mass spectrometer used was an API 5000 in ESI (-) mode.
Results and Discussion Many column chemistries were screened in order to find one capable of separating amobarbital and pentobarbital in under 20 minutes. The Kinetex 2.6 µm C18 column provided the best results separating the two isomers in under 12 minutes. A majority of comparable historical LC methods required runs > 25 mins to achieve the same resolution. Baseline resolution can be achieved with a shallower, longer gradient, but this 12 minute method allowed for accurate peak quantitation (Figure 2). Method performance, measured by recovery, RSD, and linearity over the 120 ng/ml 375 ng/ml range, confirmed great overall results. Linearity correlation figures ranged from 0.989 to 0.998, absolute recovery values ranged from 89.7 % to 102.6 %, and RSD values ranged from 0.9 to 6.7 %. (Figure 3 and Table 4). The study was repeated over 3 different days resulting in inter-assay accuracy and precision ranging from 99.1 102.1 % and 4.9-6.4 % respectively, across all analytes. Method LLQ was set at 10 ng/ml with a S/N of > 100 for all analytes.
Figure 2. LC/MS/MS Chromatogram of Barbiturates Intensity, cps 2.6e6 2.5e6 2.4e6 2.3e6 2.2e6 2.1e6 2.0e6 1.9e6 1.8e6 1.7e6 1.6e6 1.5e6 1.4e6 1.3e6 1.2e6 1.1e6 1.0e6 9.0e5 8.0e5 7.0e5 6.0e5 5.0e5 4.0e5 3.0e5 2.0e5 1.0e5 0.0 1,2 3,4 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 168 191 215 239 263 287 310 334 358 382 406 429 453 477 501 5 6 7 Sample: 1. Phenobarbital-D5 2. Phenobarbital 3. Butalbital-D5 4. Butalbital 5. Pentobarbital 6. Amobarbital 7. Secobarbital App ID 19724 min.
Figure 3. Linearity Curves Analyte Area / IS Area 1.3 1.3 1.2 1.2 1.1 1.1 1.0 1.0 0.9 0.9 0.8 0.8 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0.0 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20 1.25 Analyte Conc. / IS Conc. Analyte Area / IS Area 1.3 1.3 1.2 1.2 1.1 1.1 1.0 1.0 0.9 0.9 0.8 0.8 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0.0 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20 1.25 Analyte Conc. / IS Conc. Three point calibration curves (40 %, 100 %, 125 % of cutoff concentration) for Phenobarbital (left) and Butalbital (right)
Table 4. Recovery, RSD, and Linearity Analyte Average absolute recovery (%) RSD (n=3) Linearity R 2 Value Phenobarbital 89.7 3.5 % 0.994 Butalbital 93.2 5.5 % 0.998 Pentobarbital 100.4 3.8 % 0.989 Amobarbital 94.0 6.7 % 0.991 Secobarbital 102.6 0.9 % 0.990
Conclusion 1. The SPE extraction for amobarbital, pentobarbital, butalbital, secobarbital, and phenobarbital produced a clean extract due to the 30 % MeH wash and still resulted in high absolute recoveries. 2. The SPE workload was cut down considerably by using Strata-X- Drug N, a sorbent that requires no conditioning. 3. Quantitative resolution of the structural isomers amobarbital and pentobarbital was achieved. 4. The established method was verified to be very reproducible and rugged over a multi-day validation.
References 1..Suzuki, K. Watanabe. Drugs and poisons in humans: a handbook of practical analysis. p.306-309, 2005 2. L.K. Sørensen. Determination of acidic and neutral therapeutic drugs in human blood by liquid chromatography electrospray tandem mass spectrometry, Forensic Sci Int. 2011 Mar 20;206 (1-3):119-26 3. M.Telepchak, G. Chaney, T. August. Forensic and clinical applications of solid phase extraction. p.183-190 Trademarks Kinetex is a registered trademark of Phenomenex, Inc., in the United States, European Union, and other jurisdictions. Strata-X is a trademark of Phenomenex, Inc. Agilent is a registered trademark of Agilent Technologies, Inc. API 5000 is a trademark of AB Sciex Pte Ltd. Strata-X is patented by Phenomenex, Inc. U.S. Patent no. 7,119,145 Disclaimer Phenomenex, Inc. is not affiliated with Agilent Technologies, Inc. 2011 Phenomenex, Inc. All rights reserved.