Journal of Analytical Toxicology 2014;38:135 –142
doi:10.1093/jat/bku001 Advance Access publication February 4, 2014
Urinary Diazepam Metabolite Distribution in a Chronic Pain Population
Samantha Luk1, Rabia S. Atayee1,2, Joseph D. Ma1,2 and Brookie M. Best1,3*
1
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego (UC San Diego), 9500 Gilman Drive,
MC 0719, La Jolla, CA, USA, 2Doris A. Howell Pain and Palliative Care Service, La Jolla, CA, USA, and 3UC San Diego Department of
Pediatrics, Rady Children’s Hospital, San Diego, CA, USA
*Author to whom correspondence should be addressed. Email: brookie@ucsd.edu
Diazepam is often used as an adjuvant to pain therapy. Cytochrome
P450 (CYP) 3A4 and 2C19 metabolize diazepam into the active meta-
bolites: nordiazepam, temazepam and oxazepam. Owing to diaze-
pam’s side-effect profile, mortality risk and potential for drug –drug
interactions with CYP 3A4 and/or CYP 2C19 inhibitors, urine drug
testing (UDT) could be a helpful monitoring tool. This was a retro-
spective data analysis that evaluated urine specimens from pain
management practices for the distribution of diazepam metabolites
with and without CYP 3A4 and 2C19 inhibitors. Intersubject nordiaze-
pam, temazepam and oxazepam geometric mean fractions were
0.16, 0.34 and 0.47, respectively. Intrasubject geometric mean frac-
tions were 0.157, 0.311 and 0.494, respectively. Sex, but not age or
urinary pH, had an effect on metabolite fractions. Methadone signifi-
cantly increased temazepam and oxazepam urinary fractions via
CYP3A4 inhibition, whereas fluoxetine and esomeprazole increased
nordiazepam fractions via CYP2C19 inhibition. Although more studies
are needed, these results suggest the viability of UDT for increased
monitoring for therapy and possible drug –drug interactions.
Introduction
Diazepam is an adjuvant to opioid therapy to reduce pain-related
anxiety and to treat muscle spasms in patients with pain. It is meta-
bolized to nordiazepam by cytochrome P450 (CYP) 2C19 or to
temazepam by CYP 3A4 (1). These metabolites are biotransformed
to oxazepam by hydroxylation or demethylation (Figure 1) (2, 3).
Nordiazepam, temazepam and oxazepam are pharmacologically
active, and the latter two metabolites are commercially available
medications. Diazepam and its metabolites bind to the gamma-
aminobutyric acid (GABA) receptor to enhance GABA transmis-
sion and affect downstream processes (1, 4).
Concern is increasing over the potential for benzodiazepine de-
pendence and the difficulty of weaning patients off of benzodiaze-
pines. Diazepam has a long, variable half-life (20–50 h), which can
result in prolonged sedation, ataxia, confusion, psychoses, hypo-
tension, tremor and decreased respiratory rate. Many patients may
resist discontinuing benzodiazepines because of the strong anxio-
lytic effects, high-dependent properties and fear of withdrawal
symptoms. Withdrawal symptoms, including nausea, vomiting,
anxiety, insomnia and tremor, can occur with medication courses
as short as 2 weeks (5). When patients use diazepam with opioids,
a common practice in pain patients, the potential for overdose
and death is increased (6), and this particular combination was
the most common cause of polysubstance overdose death in the
USA from 2005 to 2009 (7). Studies conflict regarding the mech-
anism behind the interaction between benzodiazepines and
opioids. Possibly, the pharmacodynamic interaction enhances and
reinforces the reward pathway and euphoric feelings activated by
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the opioids, which can lead to increased abuse and potential over-
dose (8). Consequently, benzodiazepine monitoring is highly
encouraged.
Benzodiazepines are also susceptible to CYP-mediated drug –
drug interactions. Polymorphisms in CYP genes and drugs that
inhibit CYP have exerted clinical and pharmacokinetic effects
on diazepam and related benzodiazepines. Subjects with
increased CYP 2C19 metabolism will emerge more quickly from
general anesthesia induced by diazepam and sevoflurane (9).
CYP 3A4 and 2C19 inhibitors increase plasma concentrations of
diazepam and other benzodiazepines when co-administered
(10–13). The clinical implication of the increased plasma con-
centrations is not well characterized and can be contradictory.
For example, increased sedation has been reported when diaze-
pam or other benzodiazepines were co-administered with CYP
inhibitors (14). In contrast, some report no psychomotor dys-
function with increased plasma concentrations of benzodiaze-
pines (11, 15). Since CYP 3A4 and CYP 2C19 inhibitors are
commonly used, concurrent medication effects on diazepam dis-
position should be monitored.
Non-invasive urine testing may be preferred for monitoring di-
azepam use. Although extensive studies describe diazepam and
its metabolites in the blood (5, 16, 17), data on urinary values are
limited and conflicting. Some studies report that temazepam is
the primary urinary metabolite (18, 19), while others report that
oxazepam is the primary metabolite (20, 21). In addition, stand-
ard concentrations of diazepam metabolites in the urine are not
known (18–21). Furthermore, nordiazepam has been estab-
lished as the primary metabolite in blood (5, 16).
The aim of this retrospective data analysis was to examine
urinary distribution of diazepam metabolites in a population of
pain patients reported to be on diazepam and not any of its meta-
bolites. The study also investigated the effects of CYP 3A4 and
2C19 substrates and inhibitors on the urinary distribution of di-
azepam metabolites.
Methods
Subject selection
The study was granted Institutional Review Board-exempt status by
the University of California, San Diego Human Research Protection
Program. Specimens collected between March 2008 and May 2011
were de-identified, and 927, 772 specimens with creatinine con-
centrations .20 mg/dL were screened for inclusion.
Inclusion criteria
(i) Specimens with creatinine concentrations .20 mg/dL were
used to ensure the validity of the urine specimen (22).

Figure 1. Diazepam metabolic pathway. Diazepam is metabolized to either nordiazepam by CYP 2C19 or temazepam by CYP3A4. Nordiazepam and temazepam are hydroxylated
and demethylated to oxazepam. Oxazepam is then glucuronidated and excreted in the urine.
(ii) Physician reported that the patient was taking diazepam
and not any of the diazepam metabolites (nordiazepam,
temazepam and/or oxazepam).
(iii) The specimen had detectable concentrations of at least one
of the three diazepam metabolites. All values below the in-
strument cut-off were evaluated as zeros.
Additional inclusion criterion for intersubject analyses
(i) Single specimens from each subject’s first or only visit were
used.
Additional inclusion criterion for intrasubject analyses
(i) The subject had five or more urine specimens from differ-
ent clinic visits.
Additional inclusion criteria for the CYP substrates/
inhibitor analyses
(i) Only specimens from each subject’s first or only visit were
used.
(ii) The subject was also taking a CYP substrate or inhibitor as
defined by the Flockhart CYP Drug Interactions Table and
the FDA (23, 24).
(a) Only strong or moderate CYP3A4 inhibitors were
included. All CYP2C19 inhibitors were included
(Table I).
Specimen collection
Urine specimens were collected from physician clinics monitor-
ing patients on opioid therapy in accordance with established
guidelines (5, 25). Specimens were shipped at room temperature
by overnight courier. In some cases, urine specimens were
refrigerated until shipment. Upon arrival, liquid chromatog-
raphy– tandem mass spectrometry (LC –MS-MS) without prior
136 Luk et al.
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immunoassay screening was conducted to detect a wide variety
of drugs and drug metabolites.
Analytical procedures
An Agilent 1200 series binary pump SL Liquid Chromatography
system, well plate sampler, thermostatted column compartment,
paired with an Agilent 6410 QQQ mass spectrometer and
Agilent Mass Hunter software were used for the analysis of all
drugs. An acetonitrile –aqueous formic acid gradient ran at
0.4 mL/min. A 2.1  50 mm, 1.8 mm Zorbax SB C 18 column
was used for chromatography. The column temperature was
508C. Mobile phase A ¼ 0.1% formic acid in water and B ¼ 0.1%
formic acid in acetonitrile. The Agilent 6410 Triple Quadrupole
mass spectrometer was used in the positive electrospray ioniza-
tion mode. The nitrogen drying gas temperature was 3508C, the
flow was 12 L/min, nebulizer gas (nitrogen) pressure was 40 psi
and the capillary voltage was 3,000 V. Dwell times were 50 ms.
HPLC-grade water, acetonitrile, methanol and HPLC-grade
formic acid were obtained from VWR (Westchester, PA, USA).
Transition ions can be found in Table II.
Ion ratios for the qualifier ions were accepted if the variance
was ,20% from the target value. Deuterated internal standards
of 100 mg/mL in methanol were obtained from Cerilliant
Corporation (Round Rock, TX, USA). Calibration solutions of
nordiazepam at 40, 400, 2,560 and 5,120 ng/mL; temazepam at
50, 500, 3,200 and 6,400 ng/mL; and oxazepam at 40, 400, 2,560
and 5,120 ng/mL were prepared by diluting the standards into
synthetic urine (Microgenics Corp., Fremont, CA, USA).
Deuterated internal standards were added to calibration solutions
and to subject specimens to a final concentration of 1,200 ng/mL.
Quality control specimens of 100 and 1,000 ng/mL were placed
Table I
CYP substrates and inhibitors analyzed

CYP3A4 CYP2C19
Substrates included † Alfentanil † Felodipine † Quinidine † Amitriptyline † Nilutamide
† Alprazolam † Fentanyl † Quinine † Carisoprodol † Phenobarbitone
† Amlodipine † Finasteride † Risperidone † Citalopram † Primidone
† Aripiprazole † Gleevec † Salmeterol † Clomipramine † Progesterone
† Astemizole † Haloperidol † Sildenafil † Clopidogrel † Proguanil
† Atorvastatin † Hydrocortisone † Simvastatin † Cyclophosphamide † Propranolol

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† Buspirone † Irinotecan † Sirolimus † Hexobarbital † R-mephobarbital
† Cafergot † Lercanidipine † Sorafenib † Imipramine † S-mephenytoin
† Cerivastatin † Lidocaine † Sunitinib † Moclobemide † Teniposide
† Chlorpheniramine † Lovastatin † Tacrolimus † Nelfinavir † Warfarin
† Cilostazol † Methadone † Tamoxifen
† Cisapride † Midazolam † Taxol
† Codeine † Nateglinide † Terfenadine
† Cyclosporine † Nifedipine † Testosterone
† Dapsone † Nisoldipine † Torisel
† Dexamethasone † Nitrendipine † Trazodone
† Dextromethorphan † Ondansetron † Triazolam
† Docetaxel † Pimozide † Vincristine
† Domperidone † Progesterone † Zaleplon
† Eplerenone † Propranolol † Ziprasidone
† Estradiol † Quetiapine † Zolpidem

Inhibitors included † Aprepitant † Fluconazole † Nelfinavir † Chloramphenicol † Modafinil

† Atazanavir † Fosamprenavir † Posaconazole † Cimetidine † Omeprazole
† Boceprevir † Grapefruit juice † Ritonavir † Esomeprazole † Oxcarbazepine
† Ciprofloxacin † Imatinib † Saquinavir † Felbamate † Pantoprazole
† Clarithromycin † Indinavir † Telaprevir † Fluoxetine † Probenicid
† Conivaptan † Itraconazole † Telithromycin † Fluvoxamine † Rabeprazole
† Darunavir † Ketoconazole † Verapamil † Indomethacin † Ticlopidine
† Diltiazem † Lopinavir † Voriconazole † Ketoconazole † Topiramate
† Erythromycin † Nefazodone † Lansoprazole † Voriconazole

Table II ( pH 4.5) acetate buffer for 3 h at 458C. Samples were centri-

LC –MS-MS standards fuged for 10 min at 3,000 rpm to remove turbidity. Five microli-
ters of the specimen were injected using a CT-PAL HTS injection
Compound Transition ions Fragment Collision Dwell
(amu) voltage (V) energy (V) time (ms) device (CTC Analytics AG, Switzerland).
The interassay coefficient of variation (% CV) for all the ana-
Nordiazepam 271 ! 165 160 20 27
271 ! 140 lytes at the low and high ends of the quantification curve was
Oxazepam 287 ! 241 94 19 14 15%. At 1,000 ng/mL, the coefficients of variation for nordiaze-
287 ! 104 pam, oxazepam and temazepam were 10.6, 7.5 and 4.5%, re-
Oxazepam D5 292.1 ! 246.1 94 35 –
Temazepam 301 ! 255 120 35 73 spectively, based on the analysis of .100 runs. Similarly, at
301 ! 177 100 ng/mL, the coefficients of variation for nordiazepam, oxaze-
Temazepam D5 306 ! 206 120 35 –
pam and temazepam were 13.7, 8.5 and 6.1%, respectively. The
allowed variance for the quantitative determination in these
in each run. Upper limits of linearity were 100,000 ng/mL for all surveys is ,20%. All quantitative data were obtained from cali-
the analytes, determined by the dilution of the Cerilliant-certified bration curves with R 2 . 0.95. Most were 0.99. The lower limits
standards into synthetic urine (Microgenics negative control). of quantitation for diazepam, nordiazepam and oxazepam were
Quantitation of specimens with concentrations .100,000 ng/mL 40, 50 and 40 ng/mL, respectively.
was estimated through linear extrapolation.
Ion suppression was corrected by use of the deuterated in-
ternal standards. Specific experiments in accordance with the Statistical analysis
experiments in Pesce et al. (26) were conducted to rule out Statistical analyses were performed using OriginPro 8.5.1
large variances due to ion suppression. Efficiency of the hydroly- (OriginLab, Northampton, MA, USA) and Microsoft Excel 2010
sis procedure was determined with morphine glucuronide con- (Microsoft Corp., Redmond, WA, USA). Urine data were
trols set at 1,000 and 100 ng/mL. Morphine was used because creatinine-corrected to account for variable body mass, hydra-
this assay tests for the presence of multiple drugs that are glucur- tion status and daily water intake (27). Owing to the low urinary
onidated. Hydrolysis of the control material was considered ac- excretion of diazepam (17), metabolite fractions were used to
ceptable if the value of the recovered morphine was .90% of assess urinary diazepam metabolite excretion. The fraction of
the nominal concentration. each metabolite to the total moles of excreted diazepam meta-
Specimens were prepared for injection by incubating 25 mL of bolites and that of each metabolite to the other metabolites
urine with 50 units of b-glucuronidase Type L-II from Patella were calculated. Logarithmic values were used to approximate a
vulgata (keyhole limpet) Sigma Product number G 8132 Gaussian distribution. Two-sided two-sample t-tests were used
(Sigma-Aldrich Corp., Saint Louis, MO, USA) in 50 mL of 0.4 M to determine any statistically significant differences.

Urine Diazepam Metabolites in Pain Patients 137

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Figure 2. Fraction of diazepam metabolites for the inter-subject population. The
fraction of nordiazepam is in white, temazepam in gray and oxazepam in black. The
mean fraction of nordiazepam is 0.16, temazepam 0.34 and oxazepam 0.47 (*P ,
0.00001 for each pairwise comparison).

Results
Intersubject analysis
The log distribution of the fraction of nordiazepam, temazepam
and oxazepam from 22,509 specimens from unique subjects
approximated a Gaussian distribution (Figure 2). The geometric
mean fractions of excreted nordiazepam, temazepam and oxaze-
pam were 0.16, 0.34 and 0.47 (P , 0.00001 for each pairwise
comparison). Of the total, 92% of the population had a larger
percentage of temazepam than nordiazepam, 82% had a larger
percentage of oxazepam than nordiazepam and 71% had a
larger percentage of oxazepam than temazepam.
Most (86.8%) had all three metabolites detectable in urine
(Figure 3A and B). Approximately 13.2% did not have all three
metabolites in urine. To determine the characteristics of this
group, the data were stratified for total moles (Figure 3C). The
urine specimens of this population with one or more of the un-
detectable metabolites also had low total excreted moles.
Semi-logarithmic scatterplots of the total excreted moles of
diazepam metabolites vs. each metabolite fraction demonstrated
no relationship. A linear fit for each metabolite fraction showed
that slopes were close to zero and R 2 , 0.15. Age was not
related to metabolite concentrations for any of the diazepam
metabolites. Urinary pH and the fraction of diazepam metabolite
also demonstrated no statistically significant association.
Urinary excretion of diazepam metabolites was compared
between females (n ¼ 12,026) and males (n ¼ 9,763). The geo-
metric mean fraction of nordiazepam in females was 0.157 (95% Figure 3. Diazepam metabolites detected in urinary specimens. (A) Most of the
population on diazepam and not any of the diazepam metabolites have all three diazepam
confidence interval [95% CI] 0.155–0.158), which was 4.8% lower metabolites in the urinary specimen. However, 13.20% of the population do not make one
than those in males, 0.165 (95% CI 0.160–0.164; P , 0.01). The or more of the diazepam metabolites. Of these subjects, 90% of the subjects have
temazepam fraction in females was 0.337 (95% CI 0.334–0.339), oxazepam in the urinary specimen. (B) Most of the population makes all three
which was 7.4% higher than in males, at 0.312 (95% CI 0.309– metabolites. However, some subjects are missing one or more of the metabolites. These
subjects are located on the lines where nordiazepam, temazepam or oxazepam equal zero
0.315; P , 0.00001). The oxazepam fraction in females was 0.452
(as indicated on the plot). (C) Fractions are stratified for total moles. Subjects who are
(95% CI 0.381–0.573), 4.0% lower than the fraction in males, at missing one or more of the diazepam metabolites tend to be those that have low total
0.471 (95% CI 0.397–0.603; P , 0.00001). moles, suggesting that the subjects are close to the beginning or end of a dosing interval.

138 Luk et al.

Table III
Fractions of diazepam metabolites in urine by groups

Groups for comparison N Fraction of nordiazepam Fraction of temazepam Fraction of oxazepam

Overall populationa 21,789 0.164 (0.163, 0.165) 0.336 (0.334, 0.338) 0.468 (0.465, 0.471)
% CV 69.7 48.4 41.2
No CYP 3A4 substrates/inhibitorsa 18,218 0.167 (0.163, 0.165) 0.336 (0.335, 0.338) 0.466 (0.465, 0.471)
CYP 3A4 substratesa 3,477 0.147 (0.144, 0.150) 0.338 (0.334, 0.343) 0.481 (0.464, 0.488)
% Difference vs. control (P-value) 212.0 (P , 0.00001) þ0.6 (P . 0.05) þ3.1 (P , 0.00001)
CYP 3A4 inhibitorsa 93 0.167 (0.162, 0.193) 0.280 (0.252, 0.292) 0.530 (0.497, 0.557)

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% Difference vs. control (P-value) 0 (P . 0.05) 216.7 (P , 0.00001) þ12.1 (P , 0.05)
No CYP 2C19 substrates/inhibitorsa 18,844 0.166 (0.164, 0.167) 0.334 (0.332, 0.336) 0.468 (0.465, 0.471)
CYP 2C19 substratesa 1,175 0.161 (0.156, 0.166) 0.346 (0.339, 0.353) 0.473 (0.461, 0.484)
% Difference vs. control 23.0 (P . 0.05) þ3.47 (P , 0.05) þ1.06 (P . 0.05)
CYP 2C19 inhibitorsa 1,770 0.150 (0.145, 0.154) 0.352 (0.346, 0.359) 0.469 (0.459, 0.476)
% Difference vs. control 29.6 (P , 0.00001) þ5.1 (P , 0.00001) þ0.2 (P . 0.05)
Esomeprazolea 471 0.139 (0.137, 0.153) 0.355 (0.345, 0.371) 0.457 (0.450, 0.488)
% Difference vs. control 216.3 (P , 0.00001) þ5.9 (P , 0.00001) 21.1 (P . 0.05)
Fluoxetinea 424 0.145 (0.131, 0.149) 0.358 (0.340, 0.370) 0.469 (0.437, 0.478)
% Difference vs. control 212.7 (P , 0.00001) þ6.7 (P , 0.00001) þ0.2 (P . 0.05)
No CYP 3A4 or 2C19 substrates/inhibitorsa 16,138 0.169 (0.168, 0.171) 0.335 (0.333, 0.337) 0.465 (0.462, 0.468)
CYP 3A4 and CYP 2C19 substrate/inhibitora 36 0.183 (0.154, 0.217) 0.273 (0.237. 0.314) 0.558 (0.489, 0.626)
% Difference vs. control þ7.7 (P . 0.05) 218.5 (P , 0.005) þ16.7 (P , 0.05)

a
Geometric mean (95% CI).

Intrasubject analysis
Eight hundred and thirty-six unique subjects with 6,433 speci-
mens had an average of eight visits (range 5 –24). The geometric
mean urinary fraction of nordiazepam was 0.157 (95% CI 0.152 –
0.161; % CV ¼ 33.6). The fraction of temazepam was 0.311 (95%
CI 0.304–0.318; % CV ¼ 20.8), and that of oxazepam was 0.494
(95% CI 0.486 –0.501; % CV ¼ 18.7).
CYP substrate and inhibitor analyses
Subjects taking concurrent diazepam with CYP3A4 substrates
(n ¼ 3,477), strong or moderate CYP3A4 inhibitors (n ¼ 93), or
no CYP3A4 substrates/inhibitors (n ¼ 18,218) were compared.
Significant but very slight differences in nordiazepam and oxaze-
pam fractions were seen for those on CYP3A4 substrates.
Significant differences in temazepam and oxazepam fractions
were noted for those on CYP3A4 inhibitors (Table III). When indi-
vidual CYP3A4 substrates and inhibitors were evaluated, only sub-
jects taking methadone were significantly different from controls.
For the CYP2C19 and diazepam analyses, 1,175 subjects took
CYP2C19 substrates, 1,770 took CYP2C19 inhibitors and 18,844
were not on any CYP2C19 substrates/inhibitors. The geometric
mean fraction of temazepam was significantly different for those
on CYP2C19 substrates. The nordiazepam and temazepam frac-
tions were significantly different for those on CYP2C19 inhibi-
tors. Of the individual CYP2C19 substrates/inhibitors, only
fluoxetine and esomeprazole demonstrated significant differ-
ences from controls (Table III). Thirty-six subjects were concur-
rently on both CYP3A4 and CYP2C19 substrates and/or
inhibitors with diazepam and were compared with 16,138
control subjects not on any CYP3A4/CYP2C19 substrates/inhi-
bitors. The temazepam and oxazepam fractions were significant-
ly different in this population (Table III).
These findings support both Schwartz and Smith-Kielland’s data
on oxazepam being the major urinary metabolite of diazepam
(20, 21) but contrast with Arnold and Chiba, who found the
major metabolite to be temazepam (18, 19). The wide variation
in prior studies could be due to varied study designs and small
sample sizes (from 2 to 27 subjects), which may not represent
the general population when compared with the 22,509 subjects
in this study. Prior studies included young, healthy subjects, while
this study had a wide range of patient ages and co-morbidities. In
addition, this study utilized LC–MS-MS to quantify urine concen-
trations, whereas previous studies utilized liquid and thin-layer
chromatography. Specimens were creatinine-corrected in the
current study, which was not reported in past studies (18–21).
Although the distributions of the three metabolites overlap,
the mean fraction of each metabolite was significantly different
from each other. The % CV was greatest for nordiazepam and
least for oxazepam. The increased variability of the nordiazepam
fraction could result from the polymorphic CYP enzymes
involved in the formation of nordiazepam (10).
Even though log-transformed distributions of metabolites ap-
proximate Gaussian distributions, a group of specimens (n ¼
1,580, 7%) had an oxazepam fraction equal to 1, and contained
exclusively oxazepam. When analyzed with Grubb’s outlier test,
these data were not significant outliers. The findings were
similar whether these specimens were included or excluded.
Most of the population had all three metabolites detectable
in urine. However, 13.2% did not have one or more of the di-
azepam metabolites. When stratifying for total moles of excreted
metabolite, these specimens appear to have low total excreted
moles of metabolites, suggesting that these were collected at the
beginning or end of a dosing interval. Most were likely collected
near the end of a dosing interval due to the high number that
had detectable oxazepam.

Discussion Potential factors influencing urinary diazepam

Intersubject analysis and metabolites
In this study, oxazepam comprised the largest fraction of urinary One possible factor that could affect diazepam metabolism is the
diazepam metabolite, while nordiazepam was the smallest. total moles of excreted metabolite. However, no relationship

Urine Diazepam Metabolites in Pain Patients 139

was seen between the total moles of excreted metabolite and
the fraction of each metabolite. Although this does not preclude
an effect of diazepam load on the metabolic system, this suggests
that at therapeutic doses, the diazepam metabolic pathways are
not saturated.
Age and urinary pH do not appear to affect the fractions of
metabolites. Although kidney function decreases with age (28),
the distribution of metabolites in the urine remains unchanged.
In addition, urinary diazepam excretion does not have any acid –
base effects. Notable differences were seen, though, between
females and males. Females had higher fractions of temazepam
and lower fractions of nordiazepam and oxazepam compared
with males. Females might have increased CYP3A4-mediated di-
azepam metabolism when compared with males. In prior studies
with other drug compounds, females have demonstrated greater
CYP3A4 activity, but this observation has not been confirmed
with diazepam (29, 30). The magnitude of differences between
females and males was slight and no previous evidence has indi-
cated differential clinical effects of diazepam between sexes,
suggesting that the difference found in this study is too small to
have clinical relevance.
Intrasubject vs. intersubject analyses
The intra-subject population had a similar diazepam distribution
to the inter-subject population. The mean fractions were distinct
from each other as seen from the non-overlapping 95% CIs
between the intra-subject fractions of nordiazepam, temazepam
and oxazepam. The oxazepam fraction was the largest at 0.50
and the nordiazepam fraction was the smallest at 0.17. The simi-
larity to the inter-subject population suggests that the inter-
subject trends are also true for individual subjects. The lower
intra-subject variability in the intra-subject population suggests
that urinary diazepam excretion is more dependent on individ-
ual factors, such as CYP polymorphisms, instead of factors such
as urinary pH.
CYP substrate and inhibitor analyses
With CYP3A4 substrates, the fraction of nordiazepam was lower,
while the fraction of temazepam remained unchanged. The frac-
tion of temazepam could theoretically decrease due to competi-
tion for binding sites with concurrent administration of a
CYP3A4 substrate. However, the fraction of nordiazepam
decreased instead with a slight increase in the fraction of oxaze-
pam. Perhaps, the fraction of temazepam remained unchanged
due to the high contribution of CYP3A4 in diazepam metabol-
ism. Schmider et al. (31) postulated that CYP3A4 can constitute
60% of all diazepam metabolism in single-dose studies, and the
percentage can be even greater in clinical practice with multiple
doses. Thus, with a CYP3A4 substrate, the change in temazepam
would be negligible due to the higher affinity of diazepam for
CYP3A4. Alternatively, urinary fractions of temazepam may not
be robust enough to detect subtle perturbations in CYP3A4 ac-
tivity that would be expected with a CYP3A4 substrate. Another
possibility is that, with the increased load on CYP3A4, the body
might compensate by shunting the parent diazepam into the
CYP2C19 pathway to form nordiazepam and quickly converts
the nordiazepam into oxazepam, resulting in the small decrease
in nordiazepam and increase in oxazepam.
140 Luk et al.
On the other hand, when a strong or moderate CYP3A4 inhibi-
tor was given, the temazepam fraction was lower and the oxaze-
pam fraction higher. Since the CYP enzyme responsible for the
formation of temazepam is inhibited, the end product cannot be
formed as readily. Diazepam may be shunted into the CYP2C19
pathway to form more nordiazepam. However, the nordiazepam
fraction did not change, suggesting that nordiazepam rapidly
converts into oxazepam under conditions of CYP enzymatic
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stress. Perhaps, under high enzymatic stress, diazepam can also
engage alternative metabolic pathways to bypass CYP3A4 inhib-
ition. Minimal differences were found between individual
CYP3A4 substrates and inhibitors when compared with the
population without any concurrent CYP3A4 substrates or inhibi-
tors, except for methadone, perhaps due to higher CYP affinity
for methadone when compared with diazepam.
The minor decrease in the temazepam fraction with CYP
2C19 substrates may have been due to the increased load on
CYP 2C19. CYP 2C19 may have large capacity and can handle
the increased metabolic load of an additional CYP 2C19 sub-
strate. With CYP 2C19 inhibitors, the nordiazepam fraction
decreased and the temazepam fraction increased. When the CYP
2C19 enzyme is inhibited, less nordiazepam is made, possibly
shifting the parent diazepam compound to the CYP 3A4
pathway instead. CYP 2C19 inhibitors have the greatest inhib-
ition on the formation of temazepam in the blood when com-
pared with that of nordiazepam (32, 33), which appears to be
echoed in urine. However, as with the CYP 2C19 substrates,
changes in diazepam metabolites are minor, perhaps due to the
large capacity of the CYP 2C19 pathway. When evaluating each
individual CYP2C19 substrate and inhibitor, only fluoxetine and
esomeprazole had significant changes.
Fluoxetine and esomeprazole are potent inhibitors of
CYP2C19 (34, 35). The changes seen with concurrent fluoxet-
ine and diazepam administration shed additional light on the de-
lirium side effect observed with concurrent administration of
both drugs (10). Another interesting finding is that omeprazole
concurrently given with diazepam did not have any significant
effect on the urinary metabolite disposition, even though it is
thought to be a strong CYP 2C19 inhibitor (23), has been shown
to increase plasma concentrations of diazepam (10) and can po-
tentially inhibit the CYP 3A family (36). One possibility is that
omeprazole’s inhibition is time- and concentration-dependent,
and that maximal CYP 2C19 inhibition was not attained.
When CYP 3A4 and 2C19 substrates and/or inhibitors were
concurrently administered with diazepam, the nordiazepam
fraction did not change, the temazepam fraction decreased and
the oxazepam fraction increased. The effect seen with this par-
ticular medication pattern is similar to the effect seen with CYP
3A4 inhibitors and concurrent diazepam administration, except
that the differences are much larger with both a CYP 3A4 and a
2C19 substrate and/or inhibitor. This suggests that CYP 3A4
plays a larger role in diazepam metabolism than CYP 2C19, espe-
cially since the inhibition pattern more closely resembles that
seen with CYP 3A4 inhibitors.
The clinical implications of the shifts in urinary diazepam are
not well understood. Plasma concentrations do not directly correl-
ate with urine data in past studies. Furthermore, changes observed
in urine may be too slight to have clinical relevance, especially
since some previous studies did not observe effects on psycho-
motor function with increased benzodiazepine concentrations
(11, 15). Nevertheless, shifts in diazepam metabolism that are
observed in urine tests with CYP substrates and inhibitors can be
very helpful for clinicians, especially since the potential for
unknown adverse drug interactions with CYP substrates/inhibi-
tors is still a concern.
Limitations
One study limitation is the lack of dosage data. Variations seen in
the population may be due to external factors, differences in doses
or both. There may also be variations due to the timing of the
diazepam administration in relation to the CYP substrate/
inhibitor administration. Another limitation is the lack of plasma
data to correlate with observed urine concentrations. Urine speci-
mens were taken at variable times within the dosing regimen. A
select population may be early or late in the dosing interval, result-
ing in different metabolite distributions when compared with the
larger population. However, without definitive post-dose testing
times, whether these subjects have different diazepam metabolite
distributions due to the timing of specimen acquisition or inherent
differences in diazepam metabolism is unknown. In addition, the
medication list was reported by physicians and was not independ-
ently verified by a third party. The analysis could have included
some subjects who were not on diazepam or the studied CYP sub-
strates/inhibitors. Some subjects included in the study may have
also been on diazepam metabolites that were unreported by the
patient to the physician. Some subjects who were actually taking
diazepam may also have been excluded from the study on the basis
of the physician-reported medication. In addition, the CYP geno-
types for 3A4 and 2C19 were not known for each subject.
Although these limitations are important, several caveats also
need to be considered when interpreting these limitations.
While the dosing data for diazepam and the co-administered
medications are lacking, out of the total population analyzed,
only 13.2% of the subjects did not have all three diazepam meta-
bolites detectable in the urine (Figure 3A). Subjects without all
three compounds detected in urine would most likely consist of
subjects with urine samples drawn early in the dosing interval or
late in the dosing interval, or subjects who actually only took
one of the diazepam metabolites instead of diazepam. This popu-
lation is small compared with the larger population with urine
samples containing all three metabolites. In addition, diazepam’s
long half-life (20–50 h) may diminish the consequence of diaze-
pam administration timing, especially in the context of multiple
doses, in the metabolic pathway. Even though the CYP genotype
is unknown and has theoretical implications for diazepam me-
tabolism, there also has not yet been any data to indicate the clin-
ical relevance of CYP polymorphisms in diazepam metabolism.
Although these limitations must be taken into consideration
when interpreting the results, advantages of this study were a
large sample size and sensitive, comprehensive drug and metab-
olite assay results within each subject. In addition, the results
may reflect the type of uncontrolled situations in which clini-
cians would normally practice. Thus, despite the aforemen-
tioned limitations, the results still have practical applicability.
Conclusions
This study described urinary distribution of diazepam metabo-
lites in a large sample of pain patients. Unlike previous studies,
oxazepam was the largest fraction and nordiazepam was the
smallest fraction. These trends were seen in the majority of the
inter- and intra-subject population, suggesting that this particu-
lar pattern of diazepam excretion can potentially be extrapo-
lated to the general population. In addition, the variability in
diazepam urinary excretion may be affected more by inherent
subject-specific factors such as genetic polymorphisms in CYP
enzymes when compared with changing factors such as urinary
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pH. However, the inter- and intra-subject mean fractions of
metabolites were similar, which suggests that the current guide-
lines for diazepam administration which do not take genetics,
sex or other factors into consideration should be applicable to
most patients. Furthermore, although this study was not able to
make direct links between the urine data and plasma concentra-
tions, it was possible to observe changes in the diazepam metab-
olism as a result of co-medications. Thus, urine testing remains
viable as a potential tool, in conjunction with other medication
monitoring techniques, for clinicians to monitor their patients
for potential adverse effects. These results can serve as guide-
posts for clinicians interpreting urine test results for diazepam.
By knowing how their patient compares with the general popu-
lation, clinicians can better adjust therapy for each patient’s
needs, especially in light of the difficulty of weaning patients off
of diazepam and the potential for drug –drug interactions in
patients on multiple medications.
Acknowledgments
The authors acknowledge Amadeo J. Pesce, PhD, DABCC, for his
crucial guidance and support throughout the entirety of this
study. Urine specimens were tested and provided by Millennium
Laboratories. Dr Joseph D. Ma is a paid consultant of Millennium
Laboratories, Inc.
Funding
This work was supported in part by an educational grant pro-
vided by the University of California, San Diego Skaggs School of
Pharmacy and Pharmaceutical Sciences from an unrestricted gift
from the Millennium Research Institute (to S.L.).
References
1. Di Piero, V., Ferracuti, S., Sabatini, U., Tombari, D., Di Legge, S.,
Pantano, P. et al. (2001) Diazepam effects on the cerebral responses
to tonic pain: a SPET study. Psychopharmacology (Berlin), 158,
252–258.
2. Andersson, T., Miners, J.O., Veronese, M.E., Birkett, D.J. (1994)
Diazepam metabolism by human liver microsomes is mediated by
both S-mephenytoin hydroxylase and CYP3A isoforms. British
Journal of Clinical Pharmacology, 38, 131–137.
3. Greenblatt, D.J., Ochs, H.R., Lloyd, B.L. (1980) Entry of diazepam and
its major metabolite into cerebrospinal fluid. Psychopharmacology
(Berlin), 70, 89 –93.
4. Licata, S.C., Rowlett, J.K. (2008) Abuse and dependence liability of
benzodiazepine-type drugs: GABA(A) receptor modulation and
beyond. Pharmacology Biochemistry, and Behavior, 90, 74– 89.
5. Committee on the Review of Medicines. (1980) Systematic review of
the benzodiazepines. Guidelines for data sheets on diazepam, chlordiaz-
epoxide, medazepam, clorazepate, lorazepam, oxazepam, temazepam,
triazolam, nitrazepam, and flurazepam. Committee on the Review of
Medicines. British Medical Journal, 280, 910–912.
Urine Diazepam Metabolites in Pain Patients 141
 6. Kuryshev, Y.A., Bruening-Wright, A., Brown, A.M., Kirsch, G.E. (2010)
Increased cardiac risk in concomitant methadone and diazepam
treatment: pharmacodynamic interactions in cardiac ion channels.
Journal of Cardiovascular Pharmacology, 56, 420– 430.
7. Calcaterra, S., Glanz, J., Binswanger, I.A. (2013) National trends in
pharmaceutical opioid related overdose deaths compared to other
substance related overdose deaths: 1999–2009. Drug and Alcohol
Dependence, 131, 263– 270.
8. Jones, J.D., Mogali, S., Comer, S.D. (2012) Polydrug abuse: a review of
opioid and benzodiazepine combination use. Drug and Alcohol
Dependence, 125, 8–18.
9. Inomata, S., Nagashima, A., Itagaki, F., Homma, M., Nishimura, M.,
Osaka, Y. et al. (2005) CYP2C19 genotype affects diazepam pharma-
cokinetics and emergence from general anesthesia. Clinical
Pharmacology and Therapeutics, 78, 647– 655.
10. Desta, Z., Zhao, X., Shin, J.G., Flockhart, D.A. (2002) Clinical signifi-
cance of the cytochrome P450 2C19 genetic polymorphism. Clinical
Pharmacokinetics, 41, 913–958.
11. Kosuge, K., Jun, Y., Watanabe, H., Kimura, M., Nishimoto, M., Ishizaki, T.
et al. (2001) Effects of CYP3A4 inhibition by diltiazem on pharmaco-
kinetics and dynamics of diazepam in relation to CYP2C19 genotype
status. Drug Metabolism and Disposition: The Biological Fate of
Chemicals, 29, 1284– 1289.
12. Ozdemir, M., Aktan, Y., Boydag, B.S., Cingi, M.I., Musmul, A. (1998)
Interaction between grapefruit juice and diazepam in humans.
European Journal of Drug Metabolism and Pharmacokinetics, 23,
55 –59.
13. Zhou, S.F., Xue, C.C., Yu, X.Q., Li, C., Wang, G. (2007) Clinically im-
portant drug interactions potentially involving mechanism-based in-
hibition of cytochrome P450 3A4 and the role of therapeutic drug
monitoring. Therapeutic Drug Monitoring, 29, 687– 710.
14. Dresser, G.K., Spence, J.D., Bailey, D.G. (2000) Pharmacokinetic-
pharmacodynamic consequences and clinical relevance of cyto-
chrome P450 3A4 inhibition. Clinical Pharmacokinetics, 38, 41–57.
15. Kato, K., Yasui-Furukori, N., Fukasawa, T., Aoshima, T., Suzuki, A.,
Kanno, M. et al. (2003) Effects of itraconazole on the plasma kinetics
of quazepam and its two active metabolites after a single oral dose of
the drug. Therapeutic Drug Monitoring, 25, 473–477.
16. Greenblatt, D.J., Harmatz, J.S., Friedman, H., Locniskar, A., Shader, R.I.
(1989) A large-sample study of diazepam pharmacokinetics.
Therapeutic Drug Monitoring, 11, 652– 657.
17. Zingales, I.A. (1973) Diazepam metabolism during chronic medica-
tion unbound fraction in plasma, erythrocytes and urine. Journal of
Chromatography, 75, 55 –78.
18. Arnold, E. (1975) A simple method for determining diazepam and its
major metabolites in biological fluids: application in bioavailability
studies. Acta Pharmacologica Toxicology (Copenhagen), 36,
335–352.
19. Chiba, K., Horii, H., Chiba, T., Kato, Y., Hirano, T., Ishizaki, T. (1995)
Development and preliminary application of high-performance
liquid chromatographic assay of urinary metabolites of diazepam in
humans. Journal of Chromatography B: Biomedical Sciences and
Applications, 668, 77– 84.
20. Schwartz, M.A., Koechlin, B.A., Postma, E., Palmer, S., Krol, G. (1965)
Metabolism of diazepam in rat, dog, and man. The Journal of
Pharmacology and Experimental Therapeutics, 149, 423–435.
21. Smith-Kielland, A., Skuterud, B., Olsen, K.M., Morland, J. (2001)
Urinary excretion of diazepam metabolites in healthy volunteers and
142 Luk et al.
drug users. Scandinavian Journal of Clinical and Laboratory
Investigation, 61, 237–246.
22. Cook, J.D., Caplan, Y.H., LoDico, C.P., Bush, D.M. (2000) The charac-
terization of human urine for specimen validity determination in
workplace drug testing: a review. Journal of Analytical Toxicology,
24, 579–588.
23. United States Food and Drug Administration. (2006) Drug
Development and Drug Interactions: Table of Substrates, Inhibitors
and Inducers. US Food and Drug Administration. Silver Spring, MD.
Downloaded from https://academic.oup.com/jat/article-abstract/38/3/135/797163 by Universidad de Guadalajara user on 28 September 2018
24. Flockhart, D.A. (2007) Drug Interactions: Cytochrome P450 Drug
Interaction Table. Indiana University School of Medicine. http://
medicine.iupui.edu/clinpharm/ddis/clinical-table/ (Accessed Jul 29,
2011).
25. Washington State Agency Medical Director’s Group. (2010)
Interagency Guideline on Opioid Dosing for Chronic Non-cancer
Pain: An Educational Aid to Improve Care and Safety with Opioid
Therapy. Olympia, WA: AMDG.
26. Pesce, A., West, C., West, R., Latyshev, S., Masters-Moore, D., Friel, P.
et al. (2012) Analytical considerations when monitoring pain medi-
cations by LC–MS/MS. Journal of Analytical and Bioanalytical
Techniques S5(003), 1–55.
27. Cone, E.J., Caplan, Y.H., Moser, F., Robert, T., Shelby, M.K., Black, D.L.
(2009) Normalization of urinary drug concentrations with specific
gravity and creatinine. Journal of Analytical Toxicology, 33, 1–7.
28. Weinstein, J.R., Anderson, S. (2010) The aging kidney: physiological
changes. Advances in Chronic Kidney Disease, 17, 302– 307.
29. Tanaka, E. (1999) Gender-related differences in pharmacokinetics
and their clinical significance. Journal of Clinical Pharmacy and
Therapeutics, 24, 339– 346.
30. Scandlyn, M.J., Stuart, E.C., Rosengren, R.J. (2008) Sex-specific differ-
ences in CYP450 isoforms in humans. Expert Opinion on Drug
Metabolism and Toxicology, 4, 413– 424.
31. Schmider, J., Greenblatt, D.J., von Moltke, L.L., Shader, R.I. (1996)
Relationship of in vitro data on drug metabolism to in vivo pharmaco-
kinetics and drug interactions: implications for diazepam disposition
in humans. Journal of Clinical Psychopharmacology, 16, 267–272.
32. Jones, H.M., Hallifax, D., Houston, J.B. (2004) Quantitative prediction
of the in vivo inhibition of diazepam metabolism by omeprazole
using rat liver microsomes and hepatocytes. Drug Metabolism and
Disposition: The Biological Fate of Chemicals, 32, 572–580.
33. Zomorodi, K., Houston, J.B. (1996) Diazepam-omeprazole inhibition
interaction: an in vitro investigation using human liver microsomes.
British Journal of Clinical Pharmacology, 42, 157–162.
34. Frelinger, A.L. III., Lee, R.D., Mulford, D.J., Wu, J., Nudurupati, S.,
Nigam, A. et al. (2012) A randomized, 2-period, crossover design
study to assess the effects of dexlansoprazole, lansoprazole, esome-
prazole, and omeprazole on the steady-state pharmacokinetics and
pharmacodynamics of clopidogrel in healthy volunteers. Journal of
the American College of Cardiology, 59, 1304– 1311.
35. Jeppesen, U., Gram, L.F., Vistisen, K., Loft, S., Poulsen, H.E., Brøsen, K.
(1996) Dose-dependent inhibition of CYP1A2, CYP2C19 and
CYP2D6 by citalopram, fluoxetine, fluvoxamine and paroxetine.
European Journal of Clinical Pharmacology, 51, 73 –78.
36. Funck-Brentano, C., Becquemont, L., Lenevu, A., Roux, A., Jaillon, P.,
Beaune, P. (1997) Inhibition by omeprazole of proguanil metabolism:
mechanism of the interaction in vitro and prediction of in vivo
results from the in vitro experiments. The Journal of Pharmacology
and Experimental Therapeutics, 280, 730–738.