FORENSIC TOXICOLOGY - Bio Medical Forensics

K10 Analysis of Hydrocodone, Hydromorphone,
and Norhydrocodone in Urine Using Liquid
Chromatography - Tandem Mass
Spectrometry (LC/MS/MS)
Sandra Valtier, PhD*, Clinical Research Division, 2200 Bergquist Drive,
Building 4430, Lackland AFB, TX; Vikhyat S. Bebarta, MD, Wilford Hall
Medical Center, 2200 Bergquist Drive, Lackland AFB, TX 78236; and
Richard LaBoone, MS, Clinical Research Division, 2200 Berquist Drive,
Lackland AFB, TX 78236
The goal of this presentation is to present a validated LC/MS/MS
method for quantitative analysis of hydrocodone (HC) and metabolites
and present data from human subjects administered HC.
The presentation will impact the forensic science community by
providing data obtained from a method valdiation study of urinary HC
and its metabolites.
Measurement of HC, a semi-synthetic opioid analgesic used for
moderate and severe pain relief, can be used to monitor pain
management compliance; however, HC levels can also be useful in drug
testing cases to determine abuse or misuse of this commonly abused
opioid. Hydrocodone is metabolized to its major metabolite, HM, and to
a lesser extent to minor metabolites, NHC, and 6-α- and 6-βhydroxymetabolites.
Knowledge of metabolism and excretion profiles
of administered HC can help in determining dose, time since last dose,
and expected peak concentrations in subjects whose specific drug use is
unknown. To effectively monitor and evaluate metabolism and excretion
profiles, a sensitive and specific drug test is needed to ensure that the
drug and its metabolites can be measured to the lowest
detectable amount.
Standards spiked with concentrations of HC, HM, and NHC
ranging from 1 - 10,000 ng/mL were prepared in opioid negative urine.
Urine samples collected from subjects following HC administration were
also evaluated. The LC gradient mobile phase consisted of (A) 0.1%
formic acid and (B) acetonitrile; flow rate was set at 0.5 mL/minute. The
internal standard solution contained 1µg/mL HC-D3, HM-D3 and NHC-
D3 in methanol. A 250 µL aliquot of standard or urine was mixed with
25 µL of internal standard solution. Urine samples were hydrolyzed with
β-glucuronidase, solid phase extraction (SPE) performed, followed by 10
µL injection on the LC/MS/MS system. The mass spectrometer was set
in the ESI positive mode and analysis was performed using two multiple
reaction monitoring (MRM) transitions per analyte. The MS/MS ion
transitions monitored were m/z 300.2→199.1 and 300.2→171.0 for HC;
m/z 286.1→185.0 and 286.1→157.0 for HM; m/z 286.2→199.1 and
286.2→241.1 for NHC; m/z 303.2→199.0 for HC-D3, 289.2→185.2 for
HM-D3 and m/z 289.0→202.0 for NHC-D3.
The linear range was determined for this procedure by analysis on
six different runs on concentrations ranging from 1 to 10,000 ng/mL of
each analyte prepared in urine. The linear range was shown to be 5 to
10,000 ng/mL for HC and HM and 5 – 5,000 ng/mL for NHC with r
value > 0.99 for all compounds. The limit of detection (LOD) was 2.5
ng/mL for HC and NHC and 5 ng/mL for HM. The limit of quantitation
(LOQ) for all analytes in urine was 5 ng/mL. The method yielded good
precision with RSDs of < 10% at 100 ng/mL HC, HM, and NHC. Based
on this procedure, measurable amounts of HC, HM, and NHC were
detected in human urine for up to at least 9 hours post dose HC.
The present study will provide a validated LC/MS/MS method for
quantitation of HC, HM and NHC in urine and will also provide
evaluation of urine samples obtained from individuals administered HC.
Hydrocodone, Metabolism, LC/MS/MS
* Presenting Author
K11 Determination of Titanium Element in
Gingival Biopsies of Patients Treated With
Dental Implants by Laser Ablation –
Inductively Coupled Plasma-Mass
Spectrometry (LA-ICP-MS)
Selda Mercan, MSc, Institute of Forensic Science, Cerrahpasa, Istanbul
University, Istanbul, 34303, TURKEY; Nilufer Bolukbasi, PhD,
Department of Oral Implantology, Faculty of Dentistry, Istanbul
University, Istanbul, 34303, TURKEY; Musa K. Bolukbasi, BSc, and
Murat Yayla, BSc, Institute of Forensic Sciences, Istanbul University,
Istanbul, 34303, TURKEY; Tayfun Ozdemir, PhD, Department of Oral
Implantology, Faculty of Dentistry, Istanbul University, Istanbul, 34303,
TURKEY; and Salih Cengiz, PhD*, Institute of Forensic Sciences,
Istanbul Universitesi, Adli Bilimler Enstitüsü, CERRAHPASA TIP
FAKULTESI, Istanbul, 34303, TURKEY
After attending this presentation, attendees will understand the
transition of the titanium element into gingival biopsies which
determined using Laser Ablation-Inductively Coupled Plasma-Mass
Spectrometry (LA-ICP-MS).
This presentation will impact the forensic science community by
determining low amount of biopsy materials for any titanium element
with suitable Laser Ablation method equipped by ICP-MS.
Introduction: Titanium element is widely used material as an
implant in medical applications especially in dentistry. The use of dental
implants in the treatment of partial and complete edentulism has become
a successful treatment modality in modern dentistry. Dental implants
and their prosthetic parts are made of biocompatible materials. Today
titanium and its alloys are the first choice to fabricate implant materials.
Although titanium is a very inert material, it may corrode when in
contact with the oral cavity. If titanium corrodes it releases ions which
can cause local reactions such as pain and swelling or activate
immune response.
Materials and Methods: The study was carried out in the Clinic
of the Department of Oral Implantology at the Faculty of Dentistry and
Institute of Forensic Science, Forensic Toxicology Laboratory in
Istanbul University. The study group comprised 20 two-staged dental
implants. Osteotomy and implant installation were performed according
to the manufacturer’s surgical protocol. The implants were exposed
(second stage surgery) after three months and gingival biopsies were
collected at each site. The biopsies were stored at -18°C until use.
Samples were fixed to a lamina by an adhesive and dried in an oven at
90 o C for 2 hours.
For comparison and prediction the change of elemental composition
of gum tissues, sheep gum was used as a control matrix and confirmed
that the sheep gum had no titanium element. An adhesive material fixed
to a lamina with no sample was also used as blank for samples. Certified
Standard Material (CRM), NIST 612 glass matrix was used for quality
control sample. All samples fixed to lamina were analyzed by LA-ICP-
MS. Titanium element was detected and compared with sheep gum and
also with blank lamina.
Results and Discussion: Sheep gums were repeated five times and
the mean value was accepted as the lowest amount for Titanium element.
According to the results, some of samples showed titanium element
significantly more than sheep samples. NIST 612 glass matrix showed
that LA-ICP-MS system analyzed the titanium element close to certified
amount. Moreover, there was no response to titanium in blank lamina
which had no tissue. It can be concluded that adhesive didn’t contain any
contamination for titanium, and this may be suitable sample preparation
process for biological tissues when they are studied using
Laser Ablation.
Conclusion: Although all patients were exposed to titanium
implant for three months, elemental quantitative results were variable.
The best way to determine these kinds of patients might be monitoring
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