Investigation of the Effects of Solution Composition and Container ...

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Chemistry Department, Michigan State University, East Lansing, Michigan ... Neal A. Siegel, Robert W. Johnson, Jr., L~szl6 Litauszki, Lawrence Salvati, Jr., ...
Journal of Analytical Toxicology, Vol, 20, September 1996

Investigation of the Effectsof Solution Composition and Container Material Type on the Lossof 11-nor-Ag-THC9-Carboxylic Acid Kenneth D.W. Roth

Chemistry Department, Michigan State University, East Lansing, Michigan 48824-1322 Neal A. Siegel, Robert W. Johnson, Jr., L~szl6 Litauszki, Lawrence Salvati, Jr., Charles A. Harrington, and Larry K. Wray

Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064-3500 t Abstrac t

Materials

The loss of 11-nor-A%tetrahydrocannabinol-9-carboxylicacid (THC-COOH) from solution was studied usingfluorescence polarization immunoassay(FPIA) technology and x-ray photoelectron spectroscopy (XPS). Several materials (glass, silylated glass,high density polyethylene, polypropylene, polystyrene, polymethylmethacrylate, Teflon| and Kynar~) were studied along with three solvents (water, urine, and Abbott| cannabinoids diluent). THC-COOH lossesranging from 0 to 9.7 ng/cm2 and concentration reductions to 46% of starting values were measured. XPS indicated the presence of fluorine-labeled THC-COOH at materials surfaces. A half-life of 10 min was calculated for THC-COOH lossfrom urine stored in high density polyethylene at room temperature. Sample handling lossesduring pipetting were determined and ranged from 1.1 to 7.9 ng per aliquot. The effects of sample volume and sample handling on the THC-COOH concentrations of controls were also investigated.

Stock solutions of THC-COOH in methanol (50 IJg/mL)were obtained from Sigma Chemical (St. Louis, MO). Treated, normal human urine; cannabinoids diluent; X SystemsTM Cannabinoids Reagent Packs; AxSYM| Cannabinoids Reagent Packs; X Systems Cannabinoids Calibrators; and X Systems Multiconstituent Controls for Abused Drug Assays (MCC8s) were obtained from Abbott Laboratories (Abbott Park, IL). Treated, normal human urine was prepared by filtration, adjustment to pH 5.9, and addition of 0.1% sodium azide.

Introduction

The phenomenon of cannabinoid loss from solution during sample handling and storage has been reported previously (1-10), and it has frequently been attributed to adsorption by the material surfaces (1,5-8). Although studies have detailed the extent of cannabinoid loss from solutions to different materials (1,2,6,7), they have not reported the loss as a function of container surface area. Reporting solution loss as a function of surface area would allow prediction of loss for different containers, ll-nor-Ag-Tetrahydrocannabinol-9-carboxylic acid (THC-COOH), the major metabolite of Ag-tetrahydrocannabinol found in urine (11), was the cannabinoid studied for this communication. This communication quantitates THC-COOH loss for several materials as a function of solvent and of container surface area. In addition, the kinetics of THCCOOH loss and the amount of THC-COOH lost during pipetting were examined. The loss of THC-COOH during a variety of conditions can be described as a function of kinetics, solvent type, and container material.

,,,

llillillil

iilllillil

Figure 1. Diagram illustrating the procedure used to determine equilibrium THC-COOH loss from solution. Steps in procedure were as follows: 1. Primary stock solution prepared. 2. Solution transferred to 36 (9 materials x 4 replicates) secondary stock solutions. 3. Solutions stood 16 h to equilibriate. 4. Solutions transferred to sample containers. 5. Solutions stood 16 h to equilibriate. 6. Secondary stock solutions and corresponding samples analyzed. 7. Decrease in concentration calculated [concentration (secondary stock) - concentration (sample)].

Reproduction (photocopying) of editorial content of this iournal is prohibited without publisher's permission.

291

Journal of Analytical Toxicology, Vol. 20, September 1996

Cannabinoids diluent was treated, normal human urine with dimethyl sulfoxide (5%), sodium chloride (0.9%), and protein (0.1%) added (12). 2,2,2-Trifluoroethylamine hydrochloride (TFEA), 1-(3-dimethytaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and sodium chloride were obtained from Aldrich Chemical (Milwaukee,WI). Sigmacote| and 2-[N-morpholino]ethane-sulfonic acid (MES)were obtained from Sigma Chemical. DiSPo| 51/4-in.borosilicate glass pipettes, 1-mL SIP| disposable serologicalpipettes, Wheaton| disposable scintillation vials, and Pyrex| media/laboratory bottles were obtained from Baxter Scientific Products (McGawPark, IL). A P-1000 Pipetrnan| and RT-200 pipette tips were obtained from Rainin Instruments (Woburn, MA). Masterflex silicone tubing (1/4-in. i.d.) was

I) THC-COOH Binding to Surface H

obtained from Cole-Parmer Instrument (Chicago, IL). Glass coverslips (12 mm) were obtained from Ted Pella,(Redding, CA).Fiolax| amber glass tubing was obtained from Schott (Bayreuth, Germany) and molded by Comar (Vineland, NJ). HiD| 9512 high-density polyethylene resin was obtained from Chevron Chemical (Orange, TX) and was molded by Comar (Buena, NJ). Escorene | polypropylene sample tubes were molded in-house with Escorene medical-grade polypropylene resin obtained from Exxon (Houston, TX). PD-701 polypropylene resin was obtained from Montel (Wilmington, DE) and molded by Nypro (Gurnee, IL). 666 DW polystyrene was obtained from Dow Chemical (Midland, MI) and molded by Courtesy Medtek (Wheeling, IL). L40 acrylic (polymethylmethacrylate) resin was obtained from Cyro (Rockaway,NJ)

II) Removal of Unbound THC-COOH H

H

III) F Labeling of THC.COOH

IV) Removal of Reagents EDC HCH2CF3

NH2CH2CF3

NH2CH2CF3

P

NH2CH2CF3

EDC

V) Analysis by XPS

Figure 2. Diagram illustrating the procedure used to detect THC-COOH at materials surfaces. X-.rayphotoelectron spectroscopy was used as the technique of analysis. 292

Journal of Analytical Toxicology, Voi. 20, September 1996

and molded by Nypro (Gurnee, IL). Teflon-S| (DuPont 954-100, Wilmington, DE) and Kynar| (Dykor 830/PVDFclear, Whitford, Frazer, PA) coating of amber glass containers was performed by Thermech Engineering (Anaheim, CA). Abbott TDxFLxTM and AxSYM| instruments were used for fuorescence polarization immunoassay (FPIA) analysis of THC-COOH solutions. Surface chemical compositions were determined by x-ray photoelectron spectroscopy (XPS) measurements performed using a Perkin-Elmer (Norwalk, CT) PHI 5600 spectrophotometer. The XPS source was monochromatic 350 W A] Ks x-rays, and the detector was of the hemispherical position-sensitive type with pass energies of 187.85 eV for survey spectra and 58.70 eV for multiplex spectra. The vacuum was maintained at 5 x 10-9 Torr for all XPS experiments. Water contact angle measurements were performed with a NRL C. A. Goniometer from Ram4-Hart, Inc. (model 100-00115, Mountain Lakes, NJ). Surface tension measurements were performed with a Kruss (Charlotte, NC) processor tensiometer K12 system using the Wilhelmy plate method (13). Conductivity measurements were performed with a PHB-70X Water Analyzer. An Orion| (Orion Research, Boston, MA) 520ABenchtop pH Meter equipped with an Orion 91-03 Ag/AgCIGlass pH Electrode was used for all pH measurements.

modified P-1000 Pipetman, similar to that used by Blanc et al. (7). A short piece of Masterflexsilicone tubing (2-3 cm in length) was used to connect the Pipetman to a diSPo glass pipette. The surface areas of commodities used were determined by describing the items as a series of simple geometric shapes (e.g., cylinders, spheres, and cones). The dimensions were determined from schematic diagrams (whenever possible) or from direct measurements. The total surface areas were calculated by summing the geometric regions.

Equilibrium THC-COOH lossfrom solution The procedure used for this experiment is illustrated in Figure I. Thirty-six containers composed of nine container types were tested for each solvent. Primary stock solutions (400 mL) of approximately 100 ng THC-COOH/mL in &ionized water, in normal human urine, and in cannabinoids diluent were prepared. Thirty-six secondary stock solutions (one for each sample) were prepared by pouring approximately 8 mL of the primary stock solution into 36 scintillation vials. Secondary stock solutions were used to prevent multiple pipette transfers from a single solution. Pipetting was avoided so as not to alter the solution concentrations of THC-COOH. The secondary stock solutions were allowed to stand for at least 16 h at 2-8~ to reach equilibrium. Aliquots of the secondarystock solutions were transferred to sample containers using volumetric pipettes, and the sampleswere covered.After standing for an adMethods ditional 16 h at 2-8~ the secondarystock solutions and the sample solutions were analyzed for THC-COOH by FPIA. The General procedures filling, transfer, and analysis of all solutions were done in ranThe TDxFLxTM was used to determine the THC-COOH condomized order with secondary stocks and sample solutions centration of solutions in normal human urine or in cannabianalyzed in pairs. noids diluent. The AxSYM| was used for FPIAdetermination of The nine sample container types and the volumes used to fill THC-COOH in deionized water because the low ionic strength them are as follows: Escorene polypropylene tubes, I mL; PDwas incompatible with the TDxFLxTM. Both instruments were 701 polypropylene containers, 1 mL; polystyrene containers, 2 used in accordance with the manufacturer's instructions. mL; acrylic containers, 3 mL; glass containers, 4 mL; KynarUnless otherwise noted, THC-COOH stock solutions were coated containers, 4 mL; Teflon-S-coated containers, 4 mL; prepared by spiking solvents with 50 mg/mL THC-COOHstocks Sigmacote-treated glass containers, 4 mL; and high-density in methanol. The stock THC-COOH concentrations were polyethylene (HDPE) containers, 4 mL. Four containers of determined by FPIAand adjusted until the target concentration each type and the corresponding secondary stock solutions was reached. were analyzed. On the AxSYM,five replicates of each sample soUnless noted otherwise, solutions were transferred with a lution and secondary stock solution were analyzed. On the TDxFLx, the sample solutions were analyzed in replicates of five, but the secondary stock Table I. Equilibrium THC-COOH Loss solutions were run in replicates of four due to lack of space in the sample carousel. The Mean THC-COOH loss(ng/cm 2) THC-COOH concentration change for each Material Contact Angle Water Urine Cannab.Dil. sample was determined from the difference in mean concentrations of the sample soluChevron HiD 9512 HDPE 770 9.7• 3.8• 1.9• tion and the corresponding secondary stock Montel PD-701 Polypropylene 1070 7.2• 5.0• 1.8• solution. This value was multiplied by the Escorene Polypropylene 78o 5.7• 4.2• 1.5• solution volume and divided by the container Dow 666 DW Polystyrene 800 3.9• 3.4• 1.7• DuPont 954-I00 Teflon-S 940 4.1• 3.0• 1.2• surface area to calculate THC-COOH loss per Dykor 830/PVDF Kynar Sigmacote-Treated Glass Cyro L40 Acrylic (PMMA) Schott Fiolax Amber Glass *Not determined

-* 94o 62o 22~

2.4• 1.1• 0.8• 0.0•

1.1• 1.1• 0.9• 0.9•

0.0• 0.8• 0.6• 0.3•

cm 2.

Detection of THC-COOH usingXPS A new method of THC-COOH detection was devised.THC-COOHwas fluorine-labeled by reaction with 2,2,2-trifluoroethylamine 293

Journal of Analytical Toxicology, Vol. 20, September 1996

hydrochloride (TFEA) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC). After labeling, the fluorinelabeled THC-COOH was detected by XPS. The method is illustrated in Figure 2. A 200 ng/mL solution of THC-COOH was prepared by diluting 100 IJL of a 50 IJg/mL methanolic THC-COOH stock with 25 mL deionized water. Aliquots of the THC-COOH solution were transferred to the containers using a serological pipette. The commodities and volumes used are as follows: polypropylene container, 1 mL; acrylic container, ] mL; polystyrene container, 1 mL; HDPE container, 1 mL; glass coverslip, 75 pL; and coverslip treated with Sigmacote, 265 pL. A control was prepared for each sample by transferring an equivalent volume of deionized water to a second commodity of each type. The samples were covered and allowed to stand in contact with the solutions for 3 h at room temperature. The solutions were then discarded, and the samples were rinsed once with a buffer solution (0.5M MES, 0.5M NaC1,pH 5) (7) to remove unbound THC-COOH. The rinse volumes were the same as the solution volumes used. Aliquots of a derivatizing solution (0.1M TFEA, 0.02M EDC, 0.5M MES, 0.5M NaCl, pH 5) were applied to label surface-bound THC-COOH with fluorine. The volumes of the derivatizing solution were the same as the volumes used previously. The derivatizing solution was allowed to react for 30 min before it was discarded. The derivatization step was repeated two more times for 30 min at a time. Each sample was then rinsed twice with the buffer solution and once with deionized water. When the samples had dried, they were analyzed by XPS to determine the surface

atomic concentrations. The fluorine atomic concentrations of the controls were subtracted from the fluorine atomic concentrations of the samples to give corrected fluorine atomic concentrations. Kinetics of THC-COOH loss

A 100 ng/mL THC-COOH solution in urine was prepared and allowed to stand at least 16 h at 2-8~ The solution was then removed from the refrigerator and allowed to equilibrate for 2 h at room temperature (T = 23~ A 4-mL aliquot was transferred to a HDPE container using a volumetric pipette. The solution was allowed to stand at room temperature in the container for a period of time between 0 and 2 h to allow THCCOOH loss. The stock and sample solutions were then sampled and analyzed for THC-COOH by FPIA (five replicates each). A 60 ng/mL THC-COOH stock solution in urine was used to investigate the loss kinetics at 5.5~ The primary stock solution was divided into 10 scintillation vials, which each held approximately 10 mL solution. These portions were used as secondary stock solutions. These solutions were allowed to stand at 2-8~ for at least 16 h. Aliquots (4 mL) were transferred at 5.5~ from each secondary stock solution to the corresponding HDPE container. Loss was allowed to occur at 5.5~ for a period of time between 0 and 5 h. Aliquots from the secondary stock solutions and the sample solutions were analyzed by FPIA (five replicates each). Investigation of concentration stability

A 4-mL aliquot of a 70 ng/mL THC-COOH solution in urine

Materials

Schott Fiolax Amber Glass Cyro L40 Acrylic (PMMA) Sigmacote-Treated Glass

Solvents

Dykor 830/PVDF Kynar

9 Cannab. Dil. 9 Urine 9 Water

DuPont 954-100 Teflon-S Dow 666 DW Polystyrene Exxon Escorene Medical-Grade Polypropylene Montel PD-701 Polypropylene Chevron HiD 9512 HDPE m

3

4

5

6

7

THC-COOH loss (n~cm 2)

Figure 3. Plot of the amount of equilibrium THC-COOH

294

lost from solution as a function of solvent and material type.

8

9

10

Journal of Analytical Toxicology,Vol, 20, September1996

was transferred into a glass container.Approximately400 pL was removed and analyzed on the TDxFLxTM (five replicates). The container was covered and the solution was allowed to stand at room temperature (approximately23~ for 5 h.Another 400 pL was removedand analyzed.The vial was cappedand placed in the refrigerator (2-8~ for 8 days. The solution was removed from

the refrigerator and allowedto equilibrate at room temperature. Then, 400 IJL was removed and analyzed on the TDxFLx. Loss of THC-COOH during pipetting Three 4-mL aliquots of 110 nglmL THC-COOH solution in water and three 4-mL aliquots of a 70 ng/mL solution in urine were transferred into six glass containers. Three vials of Abbott Cannabinoids Calibrators at 80 ng/mL were also obtained. Each set of solutions (water, urine, and cannabinoids diluent) was treated in the following manner: approximately 500 mL was removed from each sample using a modified P-1000 Pipetman with a glass Pasteur pipette. The concentrations were then determined by FPIA with multiple replicates (water and urine, six replicates; cannabinoids diluent, eight replicates). The solutions were pipetted in the following manner: approximately i mL was taken up in a new pipette, and the solution was held for 15 s before being returned to the original container. After 10 pipettings in this manner, an aliquot (approximately 500 IJL) was withdrawn and the concentration of the solution was determined. The pipetting procedure was repeated twice, and the concentration of the solution was determined after 20 and 30 pipettings. The entire procedure was repeated using all combinations of solvent (water, urine, and diluent) and pipette type (glass, Sigmacote-treated glass, and polypropylene). The actual pipetting volumes were determined by weighing the solutions transferred and dividing by the solution densities. The concentration decrease at 10, 20, and 30 pipettings was determined by subtracting the solution concentration from the previous concentration. The amount of THC-COOH lost was calculated by multiplying the concentration decrease by the solution volume. The THC-COOHloss per aliquot was determined by averaging the losses and dividing by

Table II. Characteristics of THC-COOH Solvents Characteristic SurfaceTension(mN/m) Conductivity (S) pH Protein Conc. (rag/L)* DMSO Conc. (g/L)t

Water

Urine

Cannab.Dil.

71.04 t.0 x 10-~ 5.7 0 0

55.23 11,0 x 10-3 5.9 30-130 0

52.48 21.4 x 10-3 6.2 1000 50

* Seereferences12 and 16. "i" Seereference12.

Table III. THC-COOH Detection Using XPS Atomic Conc. (AC) of Fluorine Sample

Chevron HiD 9512 HDPE Montel PD-701 Polypropylene Glass Coverslip Cyro L40 Acrylic (PMMA) Dow 666 DW Polystyrene Sigmacote-keatedGlass

Samples

Controls

Corrected

0.62 0.72 0.29 0.26 0.81 0.00

0.19 0.52 0.21 0.23 0.86 0.00

0.43 0.20 0.08 0.03 0.00 0.00

Limit of Detection

Materials

Dow 666 DW Polystyrene Sigmacote-Treated Glass Cyro L40 Acrylic (PMMA)

I

Schott Fiolax Amber Glass Montel PD-701 Polypropylene Chevron HiD 9512 HDPE

IIIIIIIIIIIIIIIIIHIIIIIIIIIIIIlllfllllllllllllfl ~ |~ H~ H~ H~ HH~m ~i~ i~ H~ i~ HHl~ IIIIlflllmlllHIIlllllllHIIIIIIIIIIllfllllllH I

0.00

0.05

0.10

I

I

'

i

,

,

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Corrected fluorine atomic concentration Corrected AC = AC (Sample)- AC (Control) Figure 4. Plot of the corrected fluorine atomic concentration for the surfacesof several materials. The corrected fluorine atomic concentration determined by x-ray photoelectron spectroscopy was used as an indicator of the relative amount of THC-COOH present at the surface after reaching equilibrium with a solution in water.

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Journal o f Analytical Toxicology, Vol, 20, S e p t e m b e r 1996

10. The loss per surface area was calculated by dividing the loss per aliquot by the pipette surface area. Investigation of concentration decrease in controls

A multiconstituent control for abused drugs (MCC8) at the medium level (THC-COOHconcentration between 80 and 120 ng/mL) was removed from the refrigerator and allowed to reach room temperature. Eighteen atiquots of approximately 250 pL each were removed with a new diSPo pipette each time. Each aliquot was taken from the top of the solution and placed in a random position on the sample carousel. The aliquots were analyzed by the TDxFLxTM to determine the concentration as a function of the aliquot number. In a separate experiment, the effects of sample volume and handling were studied. Three unopened MCC8s at the medium Table IV. Kinetics of T H C - C O O H

Loss

T = 22.5~

T = 5.5~

1.7• 5.8• 5.5• _,

0.00 0.25 0.50 0.75 1.00 1.50 2.00 3.00 4.00 5.00

1.8• 2.7• 4.1• 3.2• 3.3• 4.3• 3.7• 4.7• 4.7• 6.1•

7.1 • 8.4 • .1.2

Resultsand Discussion Equilibrium THC-COOH loss from solution The amounts of THC-COOH lost at equilibrium as a function of the material and solvent are presented in Table I and Figure 3. The losses reported correspond to concentration decreases of up to 46% of the original concentration. All losses were less than 10 ng/cm2. The surface area ofa THC-COOH molecule was estimated to be approximately 125 A2 (8.5 A • 14.7 ~,, based on molecular modeling) (14). As a result, the minimum amount of THC-COOH required for monolayer coverage was calculated to be approximately 46 ng/cm 2. Because the observed losses were smaller than the minimum loss required for monolayer coverage, it seems likely that the surface binding occurred in a single layer. The solution and material surface appear to be competing to determine which has the greater affinity for THC-COOH. Polar materials (contact angle, < 70~ such as glass and acrylic showed the smallest losses, whereas nonpolar materials contact angle,

Mean Loss (ng/mL) Time (h)

level were removed from the refrigerator (2-8~ Approximately 500 t~L of each solution was removed and divided into five replicates for analysis on the TDxFLxTM. Control #1 was then stored at 2-8~ overnight. Control solutions #2 and #3 were removed with glass serological pipettes, and 500 pL of each solution was placed back into the original vial. Control #2 was placed back into the refrigerator (2-8~ as done previously, but Control #3 was placed in the refrigerator upsidedown. The samples stood at 2-8~ for 19 h. They were removed and 500 pL of each sample was analyzed.

*Not determined

lo F I 6

{

71

81

{

0

~

e fit statistics

Curve fit statistics I

y = L i m i t ( 1 - e -kt) Limit = 7.8 _+0.5 ng/cm 2 k = 4.1 _+0.9 hr -1 ty2 = 10 + 3 min

/

/

i

0.0

os

L

1.o

i

15

P-

i

zo

Time (h)

Figure 5. Plot of THC-COOH loss from urine stored in HDPE at 22.5~ as a function of time. The curve and the associated statistics were generated by fitting the data to a first-order rate equation.

296

2

= Limit(1-e-kt) Limit= 4.8 +_0.2 ng/cm2 k= 2.2 • 0.4 hF ~

1

ty2= 19 + 2 rain

0

i 1

i 2

i 3

i 4

i 5

Time (h)

Figure 6. Plot of THC-COOH loss from urine stored in HDPE at 5.5~ as a function of time. The curve and the associated statistics were generated by fitting the data to a first-order rate equation.

Journal of Analytical Toxicology, Vol. 20, September 1996

> 70~ gave rise to the largest losses. Sigmacote-treated glass was an exception to the previous trend. Although it had a large contact angle (94~ the amount of THC-COOH loss was small. Silylation of glass has been reported to reduce Ag-tetrahydrocannabinol loss (1-2), but materials silylated with Sigmacote did not show lower THC-COOH binding. In fact, the measured losses to Sigmacote-treated surfaces were nominally greater in all cases than losses to untreated glass. Sigmacote may differ chemically from the sitylation reagents studied previously. The solvent also played a large role in the amount of THCCOOH lost. Solutions in water showed the greatest losses, and solutions in cannabinoid diluent showed the smallest losses. Table II shows characteristics of the three solvents that may have an effect on the amount of THC-COOH lost. The amount of THC-COOH loss increased when the solvent surface tension increased and when the conductivity and pH decreased. Reduced THC-COOH loss at higher pH was described by Joem (5). However, the pH differences between the solutions were so small that it is unlikely they played a significant role in the amount of THC-COOH loss. The presence of protein and other organic materials in urine and cannabinoids diluent probably played a significant role in the reduced THC-COOH losses compared with losses from deionized water. Protein binding of cannabinoids has been shown to occur to a large extent (1). Also, the presence of dimethyl sulfoxide (DMSO) in cannabinoids diluent was probably an important factor in the smaller losses from diluent than from urine. Because cannabinoids have a greater solubility in organic solvents than in water (1,15), THC-COOH is more likely to remain in cannabinoids diluent than in water or urine.

Detection of THC-COOH using XPS The results of analysis by XPS are given in Table III and Table V. Concentration Stability Time

Mean concentration(ng/mt)

Initial 5h 8 days more than 5 h

70.5 • 3.1 65.1 • 3.3 68.2 _+2.6 66.6 • 3.2

Table VI. THC-COOH Loss During Pipetting THC-COOH lossper aliquot (ng) Material Sigmacote-Treated Glass Rainin Polypropylene Borosilicate Glass

Water

Urine

Cannab.Dil.

7.9 • 1.4 2.9 • 1.3 1.1 + 1.4

3.3 + 0.9 5.4 • 1.3 1.4 • 0.6

2.3 + 0.2 3.3 + 0.4 1.1 + 0.4

THC-COOH lossper aliquot (ng/cm 2) Sigmacote-Treated Glass Rainin Polypropylene Borosilicate Glass

Water

Urine

Cannab.Dil.

0.94 + 0.17 0.39 • 0A7 0.13 • 0.16

0.38 + 0.11 0.72 • 0.18 0.15 • 0.06

0.27 • 0.03 0.44 • 0.06 0.13 • 0.04

Figure 4. The atomic concentration of fluorine was interpreted as an indication of the amount of TFEA-labeled THC-COOH at the surface. Unfortunately, the TFEA appeared to bind to the material surfaces as well. The fluorine atomic concentrations of the samples minus the fluorine atomic concentrations of the control samples were used to indicate the amount of THCCOOH at the surface. The limit of detection for this approach was estimated to be 0.10% or higher. As a result, several samples gave signals that were below the limit of detection. However, the samples that gave signals greater than the estimated detection limit were the materials that showed the greatest THC-COOH loss from water. This provides some evidence that the THC-COOH being lost from solution is adsorbing to the container surface. Kinetics of THC-COOH loss The results o.f the experiments to measure the kinetics of THC-COOH loss from urine in HDPE are given in Table IV. The data in Table IV were fitted to the first-order rate equation

Loss = Limit (1--e-kt)

Eq I

to generate the curves in Figures 5 and 6. The Limit is the amount of THC-COOH that will be lost at equilibrium, k is the first-order rate constant, and t is the time in hours. The kinetic half-lives (tl~) were calculated using the relationship tr 2= (ln2)/k

Eq 2

Half-lives of 10 and 19 rain were obtained at 22.5 and 5.5 ~ respectively. This predicts that THC-COOH loss will be 95% complete in 44 min at 22.5 ~ and it will be 95% complete in 82 rain at 5.5~ This indicates that the majority of THC-COOH loss occurs during the first 1-2 h of storage. After the solution has reached equilibrium with the container, no more loss will occur. This assertion is in agreement with the observation that THC-COOH loss does not occur during storage (7).

Investigation of concentration stability The data from this experiment are summarized in Table V. The mean concentrations at 5 h and 8 days were not statistically different at the 95% confidence level. If the concentrations at 5 h and 8 days are treated as one population (concentration at t > 5 h), the mean initial concentration is greater than the mean concentration at t of 5 h or more at the 95% confidence level. These observations are consistent with the previously presented kinetic and equilibrium THC-COOH loss data. According to the kinetic data, there should be no observed loss after the 1-2 h. The predicted THC-COOH loss during the first few hours can be calculated as follows: (Loss for urine in glass container) Predicted loss= (Surface area of glass container) Solution volume

Eq 3

Predicted loss= (0.9+ 0.8 ng/cm2)(11 cm2) = 2.8 + 2.5 ng/mL 3.5 mL Observed loss= 70.5 ng/mL-66.6 ng/mL = 3.9 ng/mL

297

Journal of Analytical Toxicology, Vol. 20, September 1996

Materials

Solvents B Cannab. Dil.1 9 Urine [] Water

Glass

Polypropylene

Sigmacote-treated glass

I

I

I

I

I

I

I

l

0

1

2

3

4

5

6

7

8

THC-COOH loss per aliquot (ng)

Figure 7. Plot of THC-COOH loss during pipetting. The results are listed as a function of solvent and pipette material type.

Table VII. Concentration within MCC8 Controls MeanTHC-COOH Conc.(ng/mL)

Samples

Top5

96.4 _+2.3 94.6 __.2.2

Bottom 5

Table VIII. Effect of Volume and Handling on MCC8 Concentration. Mean THC-COOH Concentration(ng/mt) Sample 4.0 mt 0.5 mL 0.5 mL, Inverted

Initial

Final

107.4 +_2.4 106.2 _+4.4 106.3 _+ 1.9

106.0 +_4.5 96.6 _+2.0 89.1 • 3.0

The observed loss fell within one standard deviation of the predicted loss. This shows that the data in Table [ can be used to predict approximate THC-COOH losses.

Loss of THC-COOH during pipetting The results of the pipetting experiment are given in Table VI and Figure 7. The losses reported correspond to solution THC-COOH concentration decreases ranging from 8% to 57% in one aliquot. The amount of THC-COOH loss varied con298

siderably as a function of pipette material and solvent. In general, the same trends were observed as for the equilibrium binding. One notable exception was that the loss from water was greater than the loss from urine in only one case. Also, the loss observed from water in silylated glass was greater than the loss from water in polypropylene. Although the nominal losses per aliquot appear large, when the units were converted to ng/cm 2, the losses were generally smaller than the equilibrium losses reported in Table I. Loss from water in glass was the exception to the previous statement because the equilibrium loss was 0,0 ng/cm 2. The pipetting losses were expected to be smaller than those at equilibrium because the solutions were in contact with the material for a much shorter time. Investigation of concentration decrease in controls The concentrations of THC-COOH controls have been reported to decrease during normal use (7). Two experiments were performed in order to study the problem. The first experiment was intended to determine if concentration differences exist within a single THC-COOH control solution. The results of this experiment are summarized in Table VII. The difference between the mean concentrations of aliquots 1-5 and aliquots 14-18 (18 total aliquots) was not significant at the 90% confidence level. Even a significant concentration difference of less than 2 ng/mL would not account for the concentration decreases that are often observed. As a result, we can conclude that the observed concentration decreases are not the result of concentration gradients existing in the control solution.

Journal of Analytical Toxicology, Vol. 20, September 1996

The second experiment was intended to test the effect of reduced solution volume and the effect of contact with the top of the vial on the final THC-COOHconcentration. The results are summarized in Table VIII and Figure 8. The difference between the original and fina] concentrations was not significant for the first sample (4-mL final volume). However,the second (0.5-mL volume) and third (0.5-mL volume, inverted vial) samples did show a significant decrease at the 95% confidence level. All the final concentration differenceswere significant at the 95% confidence level: the concentration of the first sample was greater than the concentration of the second sample, which was greater than the concentration of third sample. The difference between the first and second solutions suggests that storage at low sample volume contributes to the observed concentration losses. Small losses from other sources, such as pipetting losses, can be magnified when the solution volume is low. The difference in concentration between the second and third samples suggests that sample handling is another important factor in the observed concentration changes. When the third sample vial was inverted, the THCCOOH solution was exposed to surfaces that normally would have no contact with the THC-COOH solution. This exposure allowed THC-COOHto bind to the top of the glass vial and the lining of the vial cap, leading to a decrease in solution concentration. This implies that careless sample handling (storing controls on their sides or tops) may be a factor in the observed concentration drop.

Conclusion THC-COOHloss from solution can be explainedas loss during equilibrium conditions (storage)or loss during kinetic conditions (pipetting or sample handling). Equilibrium losses are affectedby the solvent, the container material type, and the exposedsurface area. Lossesas high as 9.7 ng/cm2were measured, and decreases in concentration by as much as 46% were observed. Of the materials tested, high density polyethyleneshowed the greatest losses and untreated glass showed the smallest losses. Of the solutions tested, water had the greatest losses and Abbott cannabinoids diluent had the smallest losses. However,no losses were observed after the first few hours of storage. XPS was used to detect TFEA-labeledTHC-COOH at polymer surfaces, suggesting that the THC-COOHlost from solution binds to material surfaces. The preference of THC-COOHto bind to hydrophobic materials suggests some type of hydrophobic interaction as the driving force for adsorption. The ability to extract THC-COOH from material surfaceswith ethanol (7) supports this conclusion. Losses during kinetic conditions were determined to occur on the order of minutes and were affected by the temperature (tl/2 at 22.5~ = 10 min and tl/2 at 5.5~ = 19 min). Loss during pipetting was studied, and losses from 1.1 to 7.9 ng per aliquot were calculated. Untreated glass was the best pipette material tested. When the solution volumes are low, losses from pipetting and other sources can be magnified. Care should be exercised to avoid exposing THCoCOOHsolutions to new surfaces

120 9 Initial I 9 Final

100

80 r O

e-

Q r

60

r--

=~ g

40

~J

20

4.0 mL

0.5 mL

0.5 mL, Inverted

Samples Figure 8. Plot of THC-COOH concentration changes in cannabinoid controls after various treatments. The 4.0-mL column shows the change after one sampling and storage.The 0.5-mL column shows the change after the control was stored at a low volume. The 0.5-mL inverted column shows the change after storage at a low volume with the bottle inverted.

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during sample handling. These findings represent a fairly comprehensive description of THC-COOH loss and the factors that control it. It is hoped that this information will be useful to laboratories that are seeking to limit THC-COOH losses.

Acknowledgments The authors wish to thank Dr. Robert Dubler, Daniel Schmidt, Dr. Robert Schilling, and Kristine Smith for their technical assistance and insight. Thanks are due to Mr. William Motley and Ms. Sue Widner for their support and encouragement. K.R. also wishes to acknowledge the support and encouragement of Dr. Jerry Dodgson and Dr. J. Throck Watson. This work was supported in part by the Biotechnology Predoctoral Training Program of the National Institute of General Medical Sciences, Bethesda, MD 20892, Grant No. 5 T32 GM08350.

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