Benzodiazepines are extensively misused as a result of their hypnotic-sedative properties and relative availability, and they
are often used in combination with other drugs, which can result in life-threatening conditions. There is a great need for
forensic toxicologists to develop robust analytical methods to accurately and quickly measure benzodiazepines in biological
fluids like urine in cases such as overdoses, sexual assaults, and fatalities.
(PHOTO CREDIT: DAWN POLAND/GETTY IMAGES)
Introduced in the 1960s to treat a series of medical maladies ranging from seizures, convulsions, and muscle spasms to anxiety
disorders and acute alcoholism withdrawal, benzodiazepines' sedative properties and widespread availability make this group
of drugs among the most popular controlled substances in the USA. Because they are so readily available by prescription, various
forms of benzodiazepines (there are at least 17 prescribed varieties) have also become popular illegal street drugs with catchy
names like "Zannies", "K-Cuts", and "Trangs". No matter what their name, benzodiazepines taken illegally pose a huge public
health risk. Beyond a litany of physiological and psychological effects caused by the drugs, benzodiazepines are especially
toxic when mixed with alcohol, opiates, and barbiturates because they interact with the same receptors in the body and dramatically
increase the effect of the drugs. The results are often tragic, leading to a wide variety of conditions that range from seizures
and respiratory distress to coma, cardiac arrest, and even death.1 Since there are generally three time-of-effect categories for benzodiazepines (ultra-short acting, short acting, and long
acting), it is understood that the faster the reaction and effect of the drug on an individual's metabolism, the smaller the
detection window. That poses an added challenge for clinical and forensic toxicologists to develop robust analytical methods
to quickly measure benzodiazepines in biological fluids in such instances as sexual assaults, overdoses, and fatalities.2
Figure 1: Standard chromatogram of 100 ng/mL benzodiazepine mixture, inset table details the retention time and SIM for each
of the benzodiazepines. * Diazepam (Peak 8) shows two signals, corresponding to the different Cl isotope contributions.
While solid tissue samples are studied on occasion, for human studies the three most common matrices for drug testing are
blood, urine, and saliva. Urine analysis is popular because, unlike blood, it requires no technical intervention to obtain
samples. Samples tend to be collected in controlled environments, to prevent possible substitution of real samples by the
donor. In addition, urine collected from healthy individuals is a sterile matrix, and so there are few concerns with exposing
analysts to infectious agents.
For the analyst, urine samples tend to be the cleanest biological matrix. Normal urine is approximately 95% water and so lacks
a lot of the proteins and lipid-like compounds that challenge liquid chromatography coupled to mass spectrometry (LC–MS) analysis
of blood samples. Urine does contain solutes such as urea, salts, creatinine, uric acid, enzymes, fatty acids, and hormones.
Salt concentration tends to be high, so urine is often diluted by adding water prior to analysis to avoid blockages in the
LC system or fouling of the mass spectrometer with most established methods for drugs in urine being "dilute-and-shoot" methods.
However, more recently, as target levels for analysis have become more challenging, this very simple approach is inadequate
and more sophisticated urine sample preparation methods are now more common. Dilute-and-shoot does not remove interfering
matrix components that may suppress, or enhance the MS signal. Dilution of the sample also makes it more difficult to achieve
the very low limits of quantitation required for many current drug tests. In addition, as the matrix profile varies from patient
to patient, the resulting matrix effects can also vary, causing non-reproducible quantification.
Figure 2: Calibration curve for diazepam from 1–500 ng/mL. The assay showed excellent linearity with an r2 value of 0.999.
Liquid–liquid extraction (LLE) can be used as a sample preparation technique in which an immiscible solvent is added to the
sample. This separates into two liquid phases and the analyte of interest is extracted from one of the liquid partitions.
It is inexpensive and method development time is quick. However, individual methods are long, potentially labour intensive,
difficult to automate, can involve the use of large volumes of toxic organic chemicals and large quantities of glassware,
and lack the selectivity of solid-phase extraction (SPE) methods. It can also be difficult to find a method which works for
a range of analytes (and their metabolites) so more than one extraction may be required (more time).
SPE has become a more popular method of sample preparation because it allows for robust reproducible methods and lends itself
well to automation as a result of its availability in a 96-well format. SPE helps avoid potential errors in sample preparation,
reducing re-runs and dramatically increasing productivity. As one of the most cost-effective and flexible tools within the
laboratory environment, SPE also provides efficient sample concentration and purification prior to many of today's most popular
analytical techniques, including high perfomance liquid chromatography (HPLC), LC–MS, gas chromatography (GC), and GC–MS.
SPE columns are available in a wide variety of stationary phases, including reverse phase, normal phase, ion exchange, and
mixed modes, allowing the analyst to fine-tune the selectivity of their extraction methods for a wide range of analytes.
This article presents a SPE and LC–MS method for the analysis of benzodiazepines in synthetic urine samples.
Perspectives in Modern HPLC: Michael W. Dong is a senior scientist in Small Molecule Drug Discovery at Genentech in South San Francisco, California. He is responsible for new technologies, automation, and supporting late-stage research projects in small molecule analytical chemistry and QC of small molecule pharmaceutical sciences. LATEST: Seven Common Faux Pas in Modern HPLC