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Osmolality - A Novel and Sensitive Tool for Detection of Tampering of Beverages Adulterated with Ethanol,  -Butyrolactone,
and 1,4-Butanediol,and for Detection of Dilution-Tampered Demerol Syringes
KEYWORDS:
Osmolality, Forensic Chemistry, Product Tampering,
Forensic
drug testing laboratories have validated procedures in place for dealing
with solid dosage samples and are well versed in the analysis of these
types of cases. However, liquid samples containing relatively small percentages
of low molecular weight substances can present analytical challenges -
particularly if the supporting liquid matrix is itself a complex mixture
(e.g., soda or beer). In the past, the only liquid samples submitted to
this laboratory were small dropper bottles usually found to contain dilute
solutions of LSD - a relatively trivial forensic challenge. More recently,
however, the explosion of the "Rave/Club Drug" culture has resulted
in the introduction of several different drugs and/or industrial chemicals
which are also delivered in liquid form, including -hydroxybutyric acid
(GHB) or butyrate (GHB-), Many
laboratories have already developed specific and robust methods for detection
and identification of a few of the more commonly encountered compounds,
e.g., GHB. However, there are no general methods in widespread use in
forensic laboratories that are capable of rapidly and reliability detecting
the presence of any soluble, low molecular weight compound (including
novel compounds) in aqueous solutions. For example, the GHB substitutes
4-hydroxyvalerate (4-methyl-GHB), Principles of Freezing Point Osmometry1 When a solute is dissolved in a pure solvent (e.g., water), the physical/chemical properties of the solvent are changed. The freezing point is depressed, the boiling point is elevated, the vapor pressure is lowered, and the osmotic pressure is increased [these are the so-called colligative properties.] In actual practice, therefore, one mole [gram-molecular weight] of a non-dissociating solute dissolved in 1 kg of water decreases the freezing point by 1.86oC while exerting an osmotic pressure of about 17,000 mm Hg. There is no practical method for measuring osmotic pressure, however, freezing point depression is easily measured and has thus been a clinical and analytical tool for over 50 years. A solution with a measured freezing point depression of 1.86oC would be said to have an osmolality of 1 Osmol/kg or 1000 milliosmols/kg, expressed as 1000 mOsm/kg. An osmometer is a device for extremely accurate and precise determinations of the concentration of homogeneous solutions by means of freezing-point measurement. This is typically done by supercooling the target solution to several degrees below its presumed freezing point and then mechanically inducing the sample to freeze. The heat of fusion liberated during the freezing process causes the sample temperature to rise to a temporary plateau where a liquid/solid equilibrium is briefly maintained. This equilibrium temperature is, by definition, the freezing point of the solution. Osmometers include a highly accurate and precise electronic thermometer to continuously determine sample temperature and measure the freezing point of the sample. The most common current use of osmometry is in hospital toxicology laboratories, for testing serum and urine to determine electrolyte balance, diabetic acidosis, lactic acidosis, shock, stroke, and intoxication from ethanol, methanol, isopropanol, and ethylene glycol. Osmometry is also useful for monitoring rehydration therapy for treatment of severe diarrhea or to assist in recovery after collapse from over-strenuous, dehydrating exercise (such as marathons). An Advanced 3D3 Osmometer was utilized in the present study (see additional information under Experimental). In a typical analysis, 0.25 mL of a homogeneous liquid sample is pipetted into a disposable sample cup, which is then placed into the freezing chamber maintained at -7oC. At the start of the experiment, a probe containing a thermistor and stir wire descends into the sample. Over the next minute, the sample is supercooled below its freezing point. The stir wire then vibrates, causing rapid freezing. The equilibrium temperature (i.e., the freezing point) is measured, and a microprocessor converts the freezing point to osmolality and displays the result in mOsm/kg. Since the increase in osmolality is proportional to the molality of the solution, small molecular weight substances (i.e., with molecular weights less than 100), even when present in relatively low concentrations (1 - 5 percent) will detectably alter the osmolality. This makes osmometry an ideal general screening technique for substances such as GHB, GHB-, GBL, and BD. However, "classical" drugs of abuse (cocaine, heroin, LSD, etc.) have molecular weights that are too large to noticeably effect the osmolality of typical solutions.
An Advanced 3D3 Osmometer was utilized for all osmolality experiments. Osmolality calibration standard solutions of 100 mOsm/kg and 1500 mOm/kg were utilized this study. An American Optical T/S [Total Solids] Meter was used to measure the specific gravity of the solutions in the Demerol theft case. This (hand-held) instrument measures the refractive index of a liquid and provides a visual scale for conversion to specific gravity. It has a working measurement range of 1.000 to 1.035, which is adequate to measure dilute aqueous solutions. Commercial beverages, alcoholic beverages, mouthwashes, eye drops, and breath drops were purchased locally and used without any modification. Controlled substances and other abused substances were from laboratory stocks or seized exhibits. Advanced 3D3 Osmometer Evaluation: 2 Because most forensic chemists are unfamiliar with osmometry, the following details on the Advanced 3D3 Osmometer utilized in this study are provided as background. This instrument occupies approximately one square foot of counter space and weighs 25 lbs. It is solid state, consumes 150 watts an hour during operation, and has a small volume cooling bath design that allows for calibration and analysis within 15 minutes after powering up. The calibration is stored in RAM if power is disconnected. The usable measurement range is 0 - 4000 mOsm/kg (more concentrated solutions can be measured after dilution). A full range of calibration standard solutions of known osmolality are supplied and validated by the manufacturer. The instrument uses disposable 0.25 mL cuvettes (reusable cuvettes are also available). There is no auto-carousel on this model, but higher level models and other manufacturers provide this feature (some can handle up to 30 samples per hour). A typical experiment takes 2-3 minutes start to finish, and uses 0.25 mL sample. The sample is not destroyed by the osmolality analysis, and can be thawed and reanalyzed.
Linearity A linearity study was completed using the calibration standards; results are reported in Table 1 and Figure 1.
In-Run Precision 2 Ten same lot samples of Mountain Dew and Diet Mountain Dew were run, alternating between the two types to check precision as well as carry-over. Results are given in mOsm/kg (see Table 2). The low Coefficient of Variation (C.V.) values at both ends of the measurement range demonstrate excellent reproducibility. Table 2 - Within-Run Precision
Beverage Baseline Database 2,3,4 A comprehensive osmolality beverage database was needed as the first step in investigating beverage tampering with low molecular weight psychoactive substances. 146 beverages were tested. Whenever possible, 16 - 20 oz plastic, screw cap beverages were selected, as these are the most likely to be adulterated for illicit purposes. 8 oz "energy drinks" in non-resealing metal cans were also tested. [Note: The full database of results is available as an Excel Spreadsheet for download (contact the author if interested).] Sports beverage results were interesting. Although producers of sports beverages claim their products are "isotonic" (approximately equal to serum values of 275 - 295 mOsm/kg), none of the tested beverages were actually in this "physiological range". One sports beverage had a value of 190 mOsm/kg. The remaining eleven ranged from 361 - 428 mOsm/kg. Summarized results are reported in Table 3.
Most
commercial beverages are produced at multiple locations across the country
- and in some cases, across the world. To determine the validity of using
baseline data across the U.S., several different lots of each beverage
from different bottling locations were checked. Data for Pepsi and Diet
Pepsi are reported in Table 4. The results show
some variability, but good overall consistency. However, when possible,
using a control beverage in order of preference: Same lot number / same
bottling location / same country is (slightly) preferred when analyzing
a specific beverage tampering cases. [Note: International variability
was not checked in this study, and may be significant due to different
formulations in use outside the U.S.]
Consumer Products Database 2 A sampling of mouthwashes, breath drops, and eye drops were tested to determine if osmolality might be useful for forensic cases. LSD is often dosed from small dropper bottles that originally contained eye drops or breath drops. Results are reported in Table 5. Because LSD (a very high molecular weight substance) would have minimal osmolality, the finding of a very low osmolality value for a submitted exhibit of these products would indicate probable possible substitution of a water-based fluid containing LSD for the original product. Note that to prevent swelling or shrinking of the eye, eye drops are formulated to match the osmolality of natural tears; this explains their relatively low average osmolality value versus mouthwashes and breath drops. However, even this low value is much higher than a dilute aqueous solution of LSD.
Estimated Osmolality Increases from Substances of Forensic Interest The
osmolality of an adulterated beverage will be increased above its baseline
in proportion to the concentration of the agent used and that agent's
molecular weight. Estimated osmolality values are reported in Table 6
(next page). Note that (where applicable) the presented results apply
only to the free acid form of the material. Because of the dissociation
of salt forms in solution, their actual osmolality values would be expected
to be higher, in proportion to the molecular weight and concentration
of each of the components. For example, a 10 percent solution of sodium
To determine
the effect of GBL and BD on the osmolality of beverages, 20 percent V/V
solutions of GBL and BD in distilled water were prepared. Using Mountain
Dew and Diet Mountain Dew as test beverages, each was spiked with concentrations
of GBL and BD to give final solutions ranging from 0.5 - 10 percent. The
osmolalities were measured and compared to the average beverage baseline
measurements. The results are reported in Tables 7
and 8.
Illicit use of these chemicals for recreation or for facilitation of sexual assault typically involves ingestion of 1 - 3 grams. "Dosing bottles" are usually diluted to about 30 percent of the psychoactive material; thus, a 6 mL "capful" from a "dosing bottle" contains one dosage unit. At this concentration, the "dosing bottle" solution would need to be diluted 1:5 with distilled water for testing purposes, as a 30 percent solution would exceed the osmometer's upper measurement limit. At the lower concentrations, however, the results verify that adulterating a beverage with GBL or BD even at a level of only 0.5 percent will cause a measurable increase in the osmolality. This verifies that addition of one "dose" (1 - 3 grams) from a "dosing bottle" to a 16 - 20 oz. beverage will be detectable. This is important, because dilution into beverages is a typical route of administration for purposes of sexual assault, as the beverage flavor tends to disguise the "plastic" taste of the chemical (which has been described as akin to the taste of water from a garden hose left out on a hot day). Ethanol in Soda-Type Beverages 2 Numerous reports have indicated that some high school students occasionally "spike" their lunch beverages with alcoholic beverages. We therefore investigated the effect of ethanol on beverage osmolality. One oz [30 mL] of 80 proof vodka was added to 20 oz [590 mL] bottles of Mountain Dew and Diet Mountain Dew. Vodka was selected because it has almost no odor, and it is therefore the alcoholic beverage of choice for surreptitious adulteration by underage drinkers. One oz was selected as the minimum amount of alcohol that would probably be used, as being equivalent to one mixed bar drink. Actual adulteration amounts would likely be higher. The results are as follows: Mountain
Dew: Baseline - 807 mOsm/kg, With Vodka Spike - 1174 mOsm/kg
Theft of Demerol and other controlled substances by health care professionals is a recurring problem across the U.S. In June 1989, the author (working at the toxicology lab of St. Mary's Hospital in Rochester, New York) received a call from the Drug Enforcement Administration (DEA) regarding a Demerol theft investigation. A number of patients at a local hospital were complaining that they still had pain even after receiving their Demerol injections. Toxicology studies suggested that they had not in fact received any Demerol, implying diversion/theft by a nurse or other health-care professional. Hundreds of nurses were working at any one time, and they often worked on different nursing stations. To identify a suspect, the case agent systematically switched all nurses' floor schedules over several days. This process demonstrated that the patient complaints only occurred when a certain nurse was on duty. The case involved 75 mg Demerol syringes. The agent reasoned that the Demerol was being removed and used by the nurse, and a unknown liquid placed back in the syringe for patient injection. Because no patient became ill, it was felt that the nurse was using one of four sterile solutions as the replacement. The agent wanted to know exactly which of the four solutions was being used so that he could confront the suspect from a basis of fact and thereby elicit a confession. The available solutions included two normal salines and two sterile waters. Osmolality and specific gravity testing were performed on a control (untampered) Demerol syringe solution, on a suspect (tampered) Demerol syringe solution, and on all four sterile solutions. An independent quantitative analysis on the suspect Demerol solution confirmed that it only had 3.9 mg of Demerol remaining - consistent with a single plunger removal of Demerol and refill with one of the sterile solutions. The osmolality and specific gravity results are reported in Table 8. Table 8 - Osmolality and Specific Gravity Measurements in the Missing Demerol Case
As
the results show, the specific gravity testing had limited usefulness
because it could not unambiguously differentiate between all solutions.
However, the osmolality testing demonstrated that Abbott Bacteriostatic
Saline was most likely used to refill the syringe. The observed 381
mOsm/kg result in the suspect syringe (slightly higher than the Abbott
solution), was probably due to the slight effect of the 3.9 mg of Demerol
still remaining in the solution. Upon confrontation with the evidence,
the nurse admitted her guilt. With the
Identification of Sugar-Based Beverages Substituted for Diet Beverages 2,4 The accidental or purposeful substitution of a sugar-based beverage for a diet (sugarless) beverage can be harmful to a diabetic individual. Several different lots of Pepsi and Diet Pepsi were tested to determine if it would be possible to differentiate the sugar based beverage from the diet beverage. The results are as follows: Pepsi:
711-737 mOsm/kg (n=5) Although only 11 different lots were tested, there is clearly enough difference between the two types of beverages to allow a reasonable determination of diet versus sugar-based.
Dogs and cats are very sensitive to the poisonous effects of antifreeze (which contains ethylene glycol). Fatal amounts are 1.4 mL/kg for cats and 6.6 mL/kg for dogs 5. The sweet odor and taste of ethylene glycol makes it very attractive to animals, and it is therefore a particularly insidious poison. Osmolality is a very useful initial screen for suspect solutions in that it will detect the presence of ethylene glycol (and also other alcohols) at very low levels in water. Based on ethylene glycol's molecular weight of 62.02, a 1 percent solution in water would read 161 mOsm/kg, versus a typical tap water value of approximately 3 mOsm/kg.
Water is submitted on occasion to crime laboratories. Although osmolality cannot detect the presence of large molecular weight compounds in water at low concentrations [i.e., most "classic" street drugs], it is an excellent tool to identify that a submitted solution is water. Most waters tested ranged from 0 - 8 mOsm/kg. Only high-mineral content spring waters had higher values, up to 28 mOsm/kg. Non-water solvents will not freeze and no result will be obtained. Any polar solvent mixed into water will greatly increase its osmolality. Acids and bases that have been added to the water will increase the osmolality and also give a pH change. For example, a solution of 1 mL of Chlorox [5 percent hypochlorite] in 100 mL of distilled water, has a pH of 10.5 and an osmolality of 43 mOsm/kg. A solution of 1 mL of 12N HCl in 100 mL of distilled water has a pH of 1.0 and an osmolality of 243 mOsm/kg. A 1 percent solution of ethanol in distilled water has a osmolality of 158 mOsm/kg.
With
results available within 15 minutes after plug-in, on only 0.25 mL of
sample, the Advanced 3D3 Osmometer instrument used in this study (or
any equivalent osmometer) can be easily adapted for field testing at
large concert events from police D.U.I. vans. This would allow rapid
beverage screening before submission of case samples to the crime lab.
Limitations "Date-Rape" Benzodiazepines in Solution 2 As previously mentioned, the high molecular weight of common "classic" street drugs, and their low concentration in submitted solutions, makes osmolality an ineffective screening tool for their identification. For example, a single methylphenidate (Ritalin) tablet containing 5 mg of active drug and weighing 91 mg, produced a measured osmolality of only 11 mOsm/kg when dissolved in 30 mL distilled water. Therefore, osmolality is not viable for detection of drink tampering with, e.g., flunitrazepam (Rohypnol) or other sedative benzodiazepines that are employed for drug facilitated sexual assault.
Beverages are occasionally maliciously adulterated with urine. The osmolality of an individual's urine varies widely [50 - 1400 mOsm/kg] and greatly depends on the person's degree of hydration. Urea, the compound of highest concentration in the urine, varies from 0.7 - 3.3 g/100 mL, and is a better indicator of tampering than osmolality. Although a typical random urine volume of 4 - 8 oz [118 - 237 mL] may be produced, let us assume 1 oz [30 mL] was introduced into a 50 oz pot of coffee[1480 mL]. The resulting urea levels would be 14 - 67 mg/100 mL. This is easily measured with a typical urea analysis method, which usually have a dynamic range of 2 - 212 mg/100 mL. Saliva in Beverages 3 Similarly, beverages are occasionally maliciously adulterated with saliva. Amylase, which is present in very high levels in saliva [20,000 units/100 mL], is a better indicator of beverage adulteration with saliva versus osmolality. A typical 0.5 mL "spit" volume in an 8 oz [237 mL] cup of coffee would result in a measured amylase of 422 units/100 mL. This is easily measured with an amylase method having a dynamic range of 1-200 units/100 mL. Conclusions With ever increasing case loads and limited personnel resources, crime laboratories need efficient new tools to process the disturbing increases in liquid sample submissions. Osmolality, an effective analytical tool of the hospital laboratory and food and consumer products industries, is a low cost, rapid, facile, and non-destructive screening tool for forensic chemists and toxicologists.
Special thanks to Don Wiggin from Advanced Instruments for the loan of the 3D3 osmometer, and to the Rochester Institute of Technology and Drug ID Systems for providing the samples for testing.
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