gamma-Hydroxybutyrate, Silver Salt (AgGHB): Identification of
gamma-Hydroxybutyrate (GHB) via Conversion to the Silver Salt

James V. DeFrancesco
U.S. Department of Justice
Drug Enforcement Administration
North Central Laboratory
536 S. Clark St., Suite 800
Chicago, IL 60605
[email: james.v.defrancesco -at- usdoj.gov ]

ABSTRACT: A practical method for the identification of gamma-hydroxybutyrate (GHB) via infrared analysis of the corresponding silver salt is presented. The method is facile and robust, and complements the GC/MS analysis of GHB derivatives.

KEYWORDS: gamma-Hydroxybutyrate, gamma-Hydroxybutyric Acid, GHB, Sodium Oxybate, Infrared Spectrophotometry, IR, Silver Nitrate, Precipitation, Derivatization, GC/MS, Forensic Chemistry

Introduction

The continuing abuse of gamma-hydroxybutyric acid (GHB) and gamma-hydroxybutyrate (also commonly abbreviated as “GHB”) has prompted the forensic community to develop an array of analytical methodologies to identify it in its various forms (1). As new salt forms of GHB have been encountered, forensic chemists have relied primarily on infrared spectrophotometry (IR) for identification (2-5). In conjunction with IR, derivatization of GHB (usually via silylation) with subsequent analysis by Gas Chromatography/Mass Spectrometry (GC/MS) has served as a complementary means of identification of the organic ligand (2).

The silver nitrate test has been a staple in the analytical laboratory for many years; however, its use is typically limited to the presumptive identification of simple halides and commonly encountered polyatomic ions by precipitation, often followed by a solubility test for the precipitate. This study was initiated to investigate the use the silver nitrate test as a fast and easy method to presumptively identify GHB in the field. However, the resulting silver salt precipitate (i.e., AgGHB) has proven to be quite valuable for more rigorous laboratory identification.

There are several advantages to converting GHB from a group I or II metal salt (e.g., LiGHB, NaGHB, KGHB, or Ca(GHB)₂) into AgGHB. The most immediate and practical advantage is increased stability with respect to water absorption. The silver salt is far less hygroscopic than any of its group I or II metal counterparts. This property gives the analyst a much longer time frame in which to conduct further analyses. Thus, the preparation and direct characterization of the AgGHB salt directly by IR, or by subsequent derivatization followed by GC/MS analysis, increases the specificity and accuracy of the analysis.

Experimental

Intrumentation
The IR spectra were collected by two instruments: A Nicolet 6700 FT-IR equipped with a single-bounce diamond ATR accessory, and (for KBr windows) with an ATI Mattson Genesis Series FT-IR. The GC/MS data were obtained using a Agilent 6890 GC equipped with a 5873 MSD (EI, 70 eV) and HP-5MS column (30 m long x 0.32 mm ID x 0.25 μm thickness) heated from 90 °C to 280 °C at 10 °C/minute.

Precipitation of AgGHB from Samples
The following methodology effectively yields AgGHB in acceptable purity. The procedure first precipitates all ions by addition of silver nitrate, then isolates AgGHB from the other silver salts based on solubility.

1. Place five drops of an aqueous sample or a small amount (100 - 200 mg) of a solid sample in a test tube. Add 5 drops of deionized (DI) water and 5 drops of 1 N AgNO₃ (aq) and mix well.
   
   
2. If no precipitate is formed, then there are probably no interfering anions (proceed to step 3). If a precipitate is formed, add 1 mL of DI water to test the solubility of the precipitate. If the precipitate dissolves with this additional water, then proceed to step 3. (Note that at high concentrations, AgGHB forms a precipitate that readily dissolves in excess water.) If the precipitate does not dissolve with excess water, separate the precipitate by decantation, centrifugation, or filtration. This (non-dissolving) precipitate is most likely the silver salt of a halide or a polyatomic anion. Collect the remaining (clear) liquid. Add one more drop of AgNO₃ solution to ensure that all remaining interfering ions are precipitated. If a second crop precipitate forms, repeat the above process until no further precipitation occurs. Proceed to step 3.
   
   
3. Add an equal volume of alcohol (methanol, ethanol, or isopropanol) to the recovered solution to precipitate any AgGHB. If a precipitate forms, collect it by centrifugation, and dry it under an air or nitrogen purge at ~70 °C. If no precipitate forms, add additional alcohol (you may need to add up to 2 - 3 equivalent volumes of alcohol). In solution, the AgGHB precipitate is finely divided and moves like a lyotropic liquid crystal (opalescent). A convenient mechanism to remove residual alcohol, water, and remaining organic impurities is to add ether, mix, and then decant and discard the ether. The precipitate forms initially as a white powder, but may darken over time due to heat and exposure to air. This degradation (to perhaps a silver oxide, carbonate, etc.) does not appear to affect the IR spectrum - a testament to the robustness of the technique.
   
   
4. Obtain an IR spectrum. If the spectrum appears to contain excess water (broadened bands) during a KBr pellet analysis, allow the chamber to purge with nitrogen for about 15 minutes. If the same effect is observed during an ATR analysis, allow the material to simply air dry. Both purging with nitrogen and air drying will remove the water and considerably sharpen the spectral bands.
   
   
5. An optional test to perform is direct silylation of AgGHB followed by GC/MS analysis. Derivatization can be accomplished with bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing a small amount of trimethychlorosilane (TMCS), followed by heating. To guard against the introduction of silver metal ions onto the GC column, the derivatization solution should be passed over solid NaCl to capture residual silver metal ion in the form of AgCl. The resulting solution is then analyzed by GC/MS.

Results and Discussion

Analysis of AgGHB by IR
Group I and II metal salts of GHB are notoriously hygroscopic, as evidenced by the IR spectral bands which become severely blurred upon absorption of atmospheric moisture. One of the major advantages of converting these salts into AgGHB is that the silver salt is considerably less hygroscopic than any of the common Group I and II metal salts of GHB. This property is demonstrated in Figure 1, which shows the IR spectrum of a wet AgGHB sample isolated from an actual exhibit containing both a GHB salt and GBL. Several of the bands which initially appeared blurry became much sharper as the IR chamber was purged with nitrogen (which effectively drove off the residual water). In Figure 2, the transmission IR spectrum of AgGHB (i.e., acquired as a KBr window) is compared to that of the ATR spectrum. The five step procedure described above was successfully used to convert the Na, Li, K, and Ca salts of GHB into AgGHB (see Figure 3 for the IR spectra of these four salts).

Interfering Components
There are numerous components that can potentially affect the purity of AgGHB. However, few interferences were observed. Halides and other common polyatomic anions are removed by precipitation with excess AgNO₃, prior to recovery of AgGHB (i.e., while the solution is still aqueous). Sugar, a common component in liquid GHB exhibits, had no effect on the purity of the AgGHB in controlled experiments. This absence of carry-over was most likely due to sugar’s solubility in the lower alcohols. The same is true for AgNO₃, which remains in solution upon addition of the alcohol.

As noted earlier, excessive heating and prolonged exposure to air can cause minor degradation of the AgGHB. However, this darkening does not appear to affect the IR spectrum. This is demonstrated in Figure 4, which shows that the IR spectrum of a nearly four year old sample of AgGHB is indistinguishable from that of a freshly prepared sample.

Further Use of AgGHB
Further attempts to characterize AgGHB versus the Na, Li, K, and Ca GHB salts proved to be fruitless. Techniques such as HPLC and proton NMR cannot differentiate AgGHB from other GHB salts.

Conclusions

The precipitation method described above is an effective and selective method for removal of GHB salts from solution. Interferences by halide and common polyatomic anions and components such as AgNO₃ and sugar are minimized via filtration of unwanted precipitates and washings of the target precipitate with selected solvents at key points in the isolation scheme. The major advantage of forming the silver salt of GHB is its increased stability, which in turn affords the chemist greater opportunity for further testing. Perhaps the primary benefit of this work is the increased specificity. The conversion to and characterization of GHB as the silver salt, followed by secondary derivatization and characterization with a silylating reagent, significantly increases the specificity of the analysis, and thus yields a more in-depth identification.

Acknowledgements

The author would like to thank Forensic Chemist Scott J. Tschaekofske of the Minnesota Department of Public Safety, Bureau of Criminal Apprehension, for extensive discussions concerning this topic.

References

1. Anonymous. Selected references (1992 - 2003) for forensic analysis of gamma-hydroxybutyric acid, gamma-hydroxybutyrate, gamma-butyrolactone, and related compounds. Microgram Bulletin 2004;37(3):52-55.

2. Blackledge RD, Miller MD. The identification of GHB. Microgram 1991;24(7):172-179.

3. Walker L. Identification of the potassium salt of gamma-hydroxybutyrate (GHB - K ⁺). Journal of the Clandestine Laboratory Investigating Chemists Association 1999;9(1):17-8.

4. Catterton AJ, Backstrom E, and Bozenko JS. Lithium gamma-hydroxybutyrate. Journal of the Clandestine Laboratory Investigating Chemists Association 2002;12(1):26-30.

5. Tschaekofske SJ. Analysis of gamma-hydroxybutyrate (GHB) in beverages. M.S. Thesis, Michigan State University (2000).

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Figure 1: Effect of Moisture on IR Spectrum of AgGHB (Transmission).

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Figure 2: Transmission and Reflectance IR Spectra of AgGHB.

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Figure 3a: IR/ATR Spectra of AgGHB (top), NaGHB, LiGHB, KGHB, and Ca(GHB)₂.

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Table 1: Frequencies for IR/ATR Spectral Bands of AgGHB, NaGHB, LiGHB, KGHB, and Ca(GHB)₂ (in cm^-1).

AgGHB		NaGHB		LiGHB		KGHB		Ca(GHB)2
3241		3318		3318		3113		3091
2945		2959		3227		2945		2941
2877		2942		2954		2886		1587
1552		2870		2932		2845		1544
1512		1555		2877		2777		1451
1418		1451		1574		2714		1417
1398		1405		1555		1564		1407

1349		1329		1438		1480		1312
1292		1272		1409		1452		1238
1232		1229		1357		1393		1082
1162		1156		1282		1361		1032
1049		1066		1222		1318		936
1026		1015		1167		1220		910
952		946		1092		1056		868
904		920		1055		1019		810
870		881		954		914		752
800		867		914		874		664
763		774		881		859		613
698		753		778		752		600
592		666		711		687		537
567		635		672		548
		576		581
		550		540

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Figure 3b: IR/ATR Spectra of AgGHB (top), NaGHB, LiGHB, KGHB, and Ca(GHB)₂
(Expanded View of 3500 - 2400 cm^-1).

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Figure 3c: IR/ATR Spectra of AgGHB (top), NaGHB, LiGHB, KGHB, and Ca(GHB)₂
(Expanded View of 1700 - 530 cm^-1).

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Figure 4: IR/ATR Spectra of Freshly Prepared and Aged AgGHB.

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