Analysis of Benzodiazepines in Alternate Matrices by HPLC. Overview Benzodiazepines/Class/Structure Benzodiazepine Metabolism/Elimination Biological Specimens Instrumentation Analytical Method Controls Calibration Curve Comparison Internal Standard Recoveries Cases
Benzodiazepines Bzs were first introduced in 1960 s prescribed as anxiolytics, sedatives, muscle relaxants, anticonvulsants and hypnotics. All have a benzene and a diazepine ring structure. Benzodiazepines As a class, they all have similar CNS effects. Diazepam
Benzodiazepines Antianxiety drugs - Alprazolam (Xanax) - Diazepam (Valium) - Lorazepam (Ativan) - Chlordiazepoxide (Librium) - Oxazepam (Serax)
Benzodiazepines Sedative Hypnotics - Triazolam (Halcion) - Flunitrazepam (Rohypnol) - Flurazepam (Dalmane) - Temazepam (Restoril) Sedative-Hypnotics Generic Names Trade Names Daily Dosage (mg /day) BENZODIAZEPINES Long-acting Flurazepam Dalmane 15-45 Quazepam Doral 7.5-15 Intermediate-acting Estazolam Prosom 0.5-2 Temazepam Restoril 15-45 Short-acting Triazolam Halcion 0.125-0.25 NONBENZODIAZEPINE Zolpidem Zaleplon Ambien Sonata 5-20 5-20 SEDATING ANTIDEPRESSANTS Trazodone Dyserel 25-100
Benzodiazepines Continued Anticonvulsants - Clonazepam ( Klonopin) - Clorazepate (Tranxene) - Chlordiazepoxide (Librium) Anesthetic - Midazolam (Versed)
Metabolism of Benzodiazepines Table 13-3. Metabolism of benzodiazepines α -Hydroxy metabolites Long-acting metabolites 3-Hydroxy compounds Glucuronide conjugates Chlordiazepoxide Desmethylchlordiazepoxide Clorazepate Diazepam Halazepam Prazepam N-Hydroxyethylflurazepam Demoxepam Nordazepam N-desalkylflurazepam Lorazepam Temazepam Oxazepam Flurazepam Quazepam Estazolam 2-Oxo-quazepam 3-Hydroxy derivatives Alprazolam Midazolam Triazolam α-hydroxy derivatives Drugs available for clinical use are listed in bold type. With the exception of the prodrugs clorazepat and prazepam, only the glucuronide conjugates are inactive.
Elimination rates Benzodiazepines Classification by elimination rates Rapid (t 1/2 < 6 hrs) midazolam, triazolam Intermediate (t 1/2 = 6-20 hrs) alprazolam, lorazepam, temazepam Slow (t 1/2 > 20 hrs) clonazepam, diazepam, chlordiazepoxide Long Half-Lives usually means the generation of active metabolites
Specimens Analyzed Blood Liver Gastric Contents Vitreous Urine Bile
Agilent 1100 Series HPLC 0.6mL/min Flow rate 30uL injection 25 C Column Temp Gradient : 0-9min 85:15 10-22 min 72:28 23-25 25 min 85:15 25 min run, 5 min post DAD monitoring: 200-350, 4nm step UV wavelength 240 nm HP Chemstation - Data Analysis
Benzodiazepine HPLC Method 0.5 ml of sample 0.100 ml internal standard (clobazam( clobazam) Extraction solvent: 1-chlorobutane1 0.500 ml 0.2 M sodium carbonate solution Transfer of organic layer Nitrogen dry down Reconstituted with phosphate buffer:acetonitrile injected onto a reversed-phase, phenyl column
Benzodiazepine HPLC Method Calibration Curves: 0.05-2.5mg/L (5 points) Mix A: A chlordiazepoxide, oxazepam, nordiazepam,diazepam. Mix B: B alprazolam,, 7-aminoflunitrazepam7 aminoflunitrazepam,, 7-7 aminoclonazepam, alphahydroxyalprazolam, midazolam,flunitrazepam flunitrazepam,lorazepamlorazepam
Relative Retention Times Diazepam (1.18) Temazepam (0.925) Flunitrazepam (0.933) Nordiazepam ( 0.900) Oxazepam (0.760) Chlordiazepoxide (0.630) 7-aminoclonazepam (0.293) 7-aminoflunitraze(0.923) Alpha-hydroxyalprazolam hydroxyalprazolam (0.753) Lorazepam (0.787) Clonazepam (0.834) Alprazolam (0.877)
Controls Utak Benzodiazepines- Serum Chlordiazepoxide 1.0 mg/l Oxazepam 1.0 mg/l Nordiazepam 0.5 mg/l Diazepam 1.0 mg/l In-House blood, liver, deionized H 2 O Drugs of Interest 1.0 mg/l
Control Results on Various Curves Utak Benz: Chlordiazepoxide Oxazepam Nordiazepam Diazepam In-House Liver: Alprazolam Oxazepam Diazepam Spiked Conc.: 1.0 mg/l 1.0 mg/l 0.5 mg/l 1.0 mg/l 1.0 mg/l 1.2 mg/l 1.0 mg/l 1.3 mg/l 0.5 mg/l 1.3 mg/l 1.0 mg/l 2.6 mg/l Liver Curve: Blood Curve: 1.2 mg/l 0.9 mg/l 1.3 mg/l 0.8 mg/l 1.3 mg/l 0.5 mg/l 2.6 mg/l 1.1 mg/l Spiked Conc.: Liver Curve: Blood Curve: 2.5 mg/l 3.1 mg/l 2.3 mg/l 2.5 mg/l 2.3 mg/l 1.4 mg/l 2.5 mg/l 2.1 mg/l 1.0 mg/l H 2 O Curve: 20% acceptance: 0.8-1.2 mg/l 0.8-1.2 mg/l 0.4-0.60.6 mg/l 0.8-1.2 mg/l 0.9 mg/l 1.2 mg/l 0.8 0.8 mg/l 0.7 mg/l 0.8 0.5 mg/l 0.5 mg/l 0.4 1.1 mg/l 1.1 mg/l 0.8 H 2 O Curve: 20% acceptance: 1.3 mg/l 2.0-3.0 mg/l 2.3 mg/l 1.3 mg/l 1.4 mg/l 2.5 mg/l 2.0-3.0 mg/l 1.0 mg/l 0.9 mg/l 2.0-3.0 mg/l In-House H 2 O: Spiked Conc.: Liver Curve: Blood Curve: H 2 O Curve: 20% acceptance: Alprazolam 2.5 mg/l 2.4 mg/l 1.4mg/L 1.4 mg/l 2.0-3.0 mg/l Oxazepam 2.5 mg/l 3.2 mg/l 2.3 mg/l 2.5 mg/l 2.0-3.0 mg/l Diazepam 2.5 mg/l 6.0 mg/l 2.7 mg/l 2.6 mg/l 2.0-3.0 mg/l
Internal Standard Recoveries Blood Calibrators: Peak Area: 1612-1671 1671 H2O Calibrators: Peak Area: 1695-1779 1779 Liver Calibrators: Peak Area: 471-722 722
Diazepam example A known concentration of Diazepam of 1.0 mg/l was analyzed on three curves: Blood, Liver and D.I. H2O. The area of Diazepam peak was 306.652. D.I. H2O results: corresponds to an amount of 0.901 mg/l. Liver results: corresponds to an amount of 2.109 mg/l Blood results:0.951 mg/l.
Case 1:Clonazepam Circumstances of Death: The decedent was a single 24-year year-old Caucasian female who lived with a male friend in an apartment in the South Park section of San Diego. After making comments for several days about her impending death, the depressed decedent was found sitting unresponsive in her bedroom. Medics confirmed death without resuscitative efforts. Evidence of illicit and prescription drug abuse was noted.
Tox Results: Case 1 Elisa screen positive for Cocaine metabolites, Opiates and Benzodiazepines. Benzodiazepine Quant: : Peripheral Blood Clonazepam: : Trace Detected (0.018), 7-aminoclonazepam (0.68 mg/l), Nordiazepam (0.11 mg/l) Oxazepam/Diazepam: Trace Detected
Tox Results: Case 1 (Cont d) Peripheral Blood Benzoylecgonine: : 0.31 mg/l Morphine-free: 0.09 mg/l Carisoprodol: : 4.8 mg/l Meprobamate: : 16 mg/l
Tox Results: Case 1 (Cont d) Gastric Contents Carisoprodol: : 58 mg Benzodiazepines: undetected. Determination: These results would be consistent with the use of cocaine and morphine (and/or heroin) by a person also using an excessive amount of Clonazepam, while taking diazepam, carisoprodol and alcohol.
Case 2: Alprazolam The decedent was a married 50-year year-old Caucasian female who lived with her husband in Poway. She was found unresponsive and not breathing by her husband. He called 911 was instructed to provide CPR, paramedics arrived and pronounced death moments later. Background: Prior to going to bed, the decedent had not been feeling well and complained of an upset stomach and general malaise. Before going g to bed she self medicated herself with Immodium,, Mylanta and a glass of scotch with milk to releive her upset stomach. During the early morning (0430), the decedent fell out of bed, unable to lift her, the husband was instructed by his wife to leave her on the floor and give her a pillow. The husband noted marked slurred speech, but allowed her to sleep. The husband went to watch television and at 0530 returned to awaken his wife to put her to bed. He noticed that she was cold to the touch and not breathing.
Tox Results: Case 2 Gastric contents revealed 2mg of alprazolam. Peripheral blood: Alprazolam: : 0.65 mg/l Nordiazepam 0.25 mg/l Diazepam 0.28 mg/l Ethanol was not detected.
Case 3:Diazepam The decedent was a 26-year year-old Caucasian male that was found face down in the Chollas Creek riverbed area of San Diego. The decedent was found to have apparent self inflicted incised wounds as well as alcohol and medications. Suicide type notes were also found. The area were he was found was under a freeway bridge and on the western ledge of the underpass were magazines, an empty bottle of vodka, scattered pills and razor blades. There were also apparent blood spatter that led from the upper areanear the bridge and ended at a vertical concrete barrier at the western edge of the creek. At the edge was a knife with apparent blood on the blade.
Tox Results: Case 3 Peripheral Blood: (Benzodiazepine( results) Diazepam 6.8 mg/l 7-aminoclonazepam 0.10 mg/l 7-aminoflunitrazepam <0.05mg/L Temazepam <0.05 mg/l Gastric Contents: Diazepam (1/1000 dilution): 510 mg
Tox Results: Case 3 (Cont d) Report Comments: These results would be consistent with an excessive and potentially fatal ingestion of diazepam by a person also taking dipheydramine, doxylamine, psuedoephedrine,, acetaminophen, ibuprofen, clonazepam,flunitrazepam and possibly temazepam together with alcohol.