Methodologies Collection Devices, Screening Immunoassays, Confirmatory Tests

1. Methodologies 1 January 27, 2011 Methodologies (Collection Devices, Screening Immunoassays, Confirmatory Tests) Francis M. Esposito, Ph.D., D-ABFT CENTER FOR FORENSIC SCIENCES RTI INTERNATIONAL National Laboratory Certification Program (NLCP) My name is Francis Esposito and I’m employed by RTI International in the Center for Forensic Sciences. Our group holds the contract with SAMHSA to oversee the NLCP. This presentation focuses on a general overview of methodologies used for oral fluid collection devices, screening or initial test immunoassays, and confirmatory tests. My name is Francis Esposito and I’m employed by RTI International in the Center for Forensic Sciences. Our group holds the contract with SAMHSA to oversee the NLCP. This presentation focuses on a general overview of methodologies used for oral fluid collection devices, screening or initial test immunoassays, and confirmatory tests.

2. Oral Fluid Collection Types of OF collection devices Neat OF Passive pad with timed collection Passive pad with a volume indicator Chewable pad with & without stimulation Active swabbing with & without a volume indicator Oral wash Others Methodologies 2 January 27, 2011 Let’s begin with the types of oral fluid collection devices on the market and then we’ll examine a few of these more closely. There is the neat oral fluid device and the passive pad that is simply placed in the mouth with a timed collection or with a volume indicator on the handle. There is the chewable pad with and without impregnated compounds to stimulate oral fluid secretion. There is active swabbing with a pad with and without a volume indicator. And others including an oral cavity rinse. Let’s begin with the types of oral fluid collection devices on the market and then we’ll examine a few of these more closely. There is the neat oral fluid device and the passive pad that is simply placed in the mouth with a timed collection or with a volume indicator on the handle. There is the chewable pad with and without impregnated compounds to stimulate oral fluid secretion. There is active swabbing with a pad with and without a volume indicator. And others including an oral cavity rinse.

3. Oral Fluid Collection Neat OF by expectoration (spitting) Advantages Large volume can be collected Collection time is short No conversion of drug quantitation to neat value Complete recovery of drug from device Disadvantages Unsanitary and biohazardous Subject to bacteria and yeast contamination Difficult to manipulate Split specimen Laboratory aliquot for testing Methodologies 3 January 27, 2011 Let’s examine several methods of collecting oral fluid. One method is the collection of NEAT oral fluid by expectoration or “spitting”. This is a photo of such a device that consists of a funnel that is screwed onto a glass container and the donor simply spits into the container until the required volume is obtained. The advantages of this method are as listed on this slide. The disadvantages are as listed on this slide.Let’s examine several methods of collecting oral fluid. One method is the collection of NEAT oral fluid by expectoration or “spitting”. This is a photo of such a device that consists of a funnel that is screwed onto a glass container and the donor simply spits into the container until the required volume is obtained. The advantages of this method are as listed on this slide. The disadvantages are as listed on this slide.

4. Oral Fluid Collection Collection devices with absorbent pad Pad in donor’s mouth becomes saturated with OF Removed at designated time or using volume indicator Collection paddle placed in tube containing buffer/preservative Buffer solution elutes drugs from pad Buffer solution used for laboratory drug tests Methodologies 4 January 27, 2011 Here is an example of one of several collection devices with an absorbent pad. This device contains a cellulose collection pad. The pad is placed in the donor’s mouth. The pad becomes saturated with oral fluid and is removed either at the end of a timed collection or with some devices, when the volume indicator dye moves up to the window on the handle. The collection pad is placed in a transport tube that contains a liquid buffer with a preservative. The buffer solution elutes the drugs from the pad and is used for laboratory drug testing.Here is an example of one of several collection devices with an absorbent pad. This device contains a cellulose collection pad. The pad is placed in the donor’s mouth. The pad becomes saturated with oral fluid and is removed either at the end of a timed collection or with some devices, when the volume indicator dye moves up to the window on the handle. The collection pad is placed in a transport tube that contains a liquid buffer with a preservative. The buffer solution elutes the drugs from the pad and is used for laboratory drug testing.

5. Oral Fluid Collection Collection devices with pad but no buffer solution Device uses a chewable pad Pad is placed in transport tube Tube is centrifuged to remove OF from the pad Methodologies 5 January 27, 2011 Several devices use an absorbent pad or wad that is placed in a transport container that does not have a buffer/preservative solution. This photo is an example of a chewable pad that is placed into the mouth of the donor until saturated and then placed into a transport tube. At the laboratory, the tube is centrifuged to remove the oral fluid from the pad. The pad is removed to obtain the oral fluid at the bottom of the tube for drug testing.Several devices use an absorbent pad or wad that is placed in a transport container that does not have a buffer/preservative solution. This photo is an example of a chewable pad that is placed into the mouth of the donor until saturated and then placed into a transport tube. At the laboratory, the tube is centrifuged to remove the oral fluid from the pad. The pad is removed to obtain the oral fluid at the bottom of the tube for drug testing.

6. Oral Fluid Collection Collection devices with absorbent pads Advantages No direct contact with biohazardous OF if using a paddle pad Easy manipulation of OF if eluted into buffer solution Possible drug stability in the buffer solution Disadvantages Speed of collection slow for dry mouth (smokers, drug users, viscous) Lack of uniformity among devices (time vs. indicator; buffer or none) Possible inadequate volume for multiple drugs or repeat analyses Number of variables that affect collection device performance Methodologies 6 January 27, 2011 Collection devices with absorbent pads as those in the previous two slides, have advantages and disadvantages. Some of the advantages are those listed on this slide. Some of the disadvantages are those listed on this slide.Collection devices with absorbent pads as those in the previous two slides, have advantages and disadvantages. Some of the advantages are those listed on this slide. Some of the disadvantages are those listed on this slide.

7. Oral Fluid Collection Issues with collection devices with absorbent pads Volume of OF collected Need adequate amount for multiple drug analyses Accuracy of the volume collected will impact the final drug level Ex: expect 1 mL but collects 1.5 mL then 50% higher drug level Pad absorption Pad capable of absorbing required analytes Removal of analytes from pad Studies reveal analyte recovery varies among devices Ex: amphetamine 16-97% but cocaine 61-95%* Lipophilic analyte (THC) lowest recovery <12.5-85.4%** Analyte Stability Stable at least 2 weeks in buffer or tube when refrigerated *Crouch et al. Ther Drug Monit 2008;30:188 **Langel et al. JAT 2008;32:393 Methodologies 7 January 27, 2011 Let’s look at some of the issues that affect the performance of collection devices with absorbent pads. For the volume of oral fluid, an adequate amount will need to be collected for multiple drug analyses or for reanalysis. The accuracy of the volume collected is important. As an example, if the lab analysis is based on the collection of 1 mL but the actual amount of oral fluid is 1.5 mL, the calculated drug concentration will be 50% higher. The standards set for pad devices should require limits on the variability of volume. The pads need to be evaluated for their capability to absorb the required analytes. Removal of absorbed analytes from the pad is an important issue. Scientific studies reveal analyte recovery varies among devices. For example, in the study by Crouch and others, they found recoveries varied dramatically among 6 devices. Amphetamine percent recovery had a range from 16-97% while cocaine performed better with a range of 61-95%. Langel and others performed recovery studies on 10 devices. They found THC, a lipophilic compound, to have the lowest recovery of tested analytes with some devices that performed poorly had <12.5% recovery to one with the highest recovery of 85.4%. Analyte stability is important with or without a buffer solution. Analytes should be stable at least 2 weeks when refrigerated. Let’s look at some of the issues that affect the performance of collection devices with absorbent pads. For the volume of oral fluid, an adequate amount will need to be collected for multiple drug analyses or for reanalysis. The accuracy of the volume collected is important. As an example, if the lab analysis is based on the collection of 1 mL but the actual amount of oral fluid is 1.5 mL, the calculated drug concentration will be 50% higher. The standards set for pad devices should require limits on the variability of volume. The pads need to be evaluated for their capability to absorb the required analytes. Removal of absorbed analytes from the pad is an important issue. Scientific studies reveal analyte recovery varies among devices. For example, in the study by Crouch and others, they found recoveries varied dramatically among 6 devices. Amphetamine percent recovery had a range from 16-97% while cocaine performed better with a range of 61-95%. Langel and others performed recovery studies on 10 devices. They found THC, a lipophilic compound, to have the lowest recovery of tested analytes with some devices that performed poorly had <12.5% recovery to one with the highest recovery of 85.4%. Analyte stability is important with or without a buffer solution. Analytes should be stable at least 2 weeks when refrigerated.

8. Screening Immunoassays 2 Immunoassay types for parent drugs and metabolites Heterogeneous Enzyme Immunoassays ELISA Advantages Sensitivity (1ng/mL or lower) Small sample size (25uL) Specificity (amp with no cross reaction to methamp) Disadvantages Wash or separation step Labor intensive unless automated Methodologies 8 January 27, 2011 My second topic in this presentation is screening (or initial testing) immunoassays which are assays that use antibodies to detect parent drugs and metabolites in oral fluids. Since the concentration of analytes is lower in oral fluid than urine, screening tests require greater sensitivity. There are two types of immunoassays to detect drugs and metabolites in oral fluid. The first type is a heterogeneous enzyme immunoassay. The enzyme linked immunosorbent assay (ELISA) is commonly used for screening drugs in oral fluid. Their advantages include nanogram or picogram per mL sensitivity with small sample size of 25 uL for each drug class. ELISA kits can also be very specific to analytes in a drug class. For example, there are some ELISA kits that are specific for amphetamine with essentially no cross reactivity to methamphetamine. Disadvantages. Since it is a heterogeneous assay, a wash or separation step of the bound and free antigen is required before an absorbance reading of the labelled antigen. This process can increase labor costs, but automation procedures can reduce labor costs when used for pipetting, washing and absorbance measurements.My second topic in this presentation is screening (or initial testing) immunoassays which are assays that use antibodies to detect parent drugs and metabolites in oral fluids. Since the concentration of analytes is lower in oral fluid than urine, screening tests require greater sensitivity. There are two types of immunoassays to detect drugs and metabolites in oral fluid. The first type is a heterogeneous enzyme immunoassay. The enzyme linked immunosorbent assay (ELISA) is commonly used for screening drugs in oral fluid. Their advantages include nanogram or picogram per mL sensitivity with small sample size of 25 uL for each drug class. ELISA kits can also be very specific to analytes in a drug class. For example, there are some ELISA kits that are specific for amphetamine with essentially no cross reactivity to methamphetamine. Disadvantages. Since it is a heterogeneous assay, a wash or separation step of the bound and free antigen is required before an absorbance reading of the labelled antigen. This process can increase labor costs, but automation procedures can reduce labor costs when used for pipetting, washing and absorbance measurements.

9. Screening Immunoassays Homogeneous assays Advantages Automated assays Lower cost per test Disadvantages Specificity Sensitivity challenges Methodologies 9 January 27, 2011 The second type of immunoassay for analytes in oral fluid is the homogeneous assay. These assays do not require a separation step of the bound and free antigen. Advantages for this type of assay are the assays are designed for automated procedures that permit rapid throughput and are not labor intensive that should lead to a lower cost per test. Disadvantages. Specificity can be an issue with cross reactivity of over-the-counter medications especially for the amphetamine class of drugs. Sensitivity is a challenge with homogeneous reagents in that the optical measurement is made in the presence of the biological matrix. The second type of immunoassay for analytes in oral fluid is the homogeneous assay. These assays do not require a separation step of the bound and free antigen. Advantages for this type of assay are the assays are designed for automated procedures that permit rapid throughput and are not labor intensive that should lead to a lower cost per test. Disadvantages. Specificity can be an issue with cross reactivity of over-the-counter medications especially for the amphetamine class of drugs. Sensitivity is a challenge with homogeneous reagents in that the optical measurement is made in the presence of the biological matrix.

10. Screening Immunoassays Initial test analytes and cutoff concentrations (ng/mL) Methodologies 10 January 27, 2011 This table shows the initial test analytes and cutoff concentration for oral fluid from the 2004 “Proposed Revisions to Mandatory Guidelines for Federal Workplace Drug Testing Programs” compared to initial test analytes and cutoffs for urine from the October 2010 “Revised Mandatory Guidelines for Federal Workplace Drug Testing Programs”. Notice that the initial test analytes are the same to include 6-AM and MDMA recently added to the urine panel, but cutoffs for oral fluid are less than urine due to the lower concentration of drugs and metabolites in oral fluid. Also included in this table are the manufacturers’ immunoassay kit cutoff ranges for each oral fluid analyte. As you can see the manufacturers’ cutoffs are not standardized with ranges that are as large as a 10-fold difference for THC and a kit specific to amphetamine. Some manufacturers offer separate immunoassay kits for methamphetamine and amphetamine.This table shows the initial test analytes and cutoff concentration for oral fluid from the 2004 “Proposed Revisions to Mandatory Guidelines for Federal Workplace Drug Testing Programs” compared to initial test analytes and cutoffs for urine from the October 2010 “Revised Mandatory Guidelines for Federal Workplace Drug Testing Programs”. Notice that the initial test analytes are the same to include 6-AM and MDMA recently added to the urine panel, but cutoffs for oral fluid are less than urine due to the lower concentration of drugs and metabolites in oral fluid. Also included in this table are the manufacturers’ immunoassay kit cutoff ranges for each oral fluid analyte. As you can see the manufacturers’ cutoffs are not standardized with ranges that are as large as a 10-fold difference for THC and a kit specific to amphetamine. Some manufacturers offer separate immunoassay kits for methamphetamine and amphetamine.

11. Screening Immunoassays Specimen validity testing Urine Specimens can be adulterated or substituted Creatinine, specific gravity, pH and oxidant testing required Oral Fluid Immunoglobulin (IgG) concentration should be =0.1 ug/mL Some say SVT not required due to observed collection Methodologies 11 January 27, 2011 Urine specimens can be adulterated or substituted by the donor due to the availability of adulterant agents and to the nature of the unobserved collection process. Creatinine, specific gravity, pH, and oxidant testing are required on all urine specimens under the Mandatory Guidelines of April 2004 to detect abnormal specimens. The Proposed Guidelines of 2004 recommended the determination of immunoglobulin concentration of IgG on every oral fluid specimen. Concentrations of <0.1 ug/mL of IgG would indicate a substituted specimen. Some scientists believe that specimen validity testing is not needed for oral fluid specimens due to the observed collection process. Urine specimens can be adulterated or substituted by the donor due to the availability of adulterant agents and to the nature of the unobserved collection process. Creatinine, specific gravity, pH, and oxidant testing are required on all urine specimens under the Mandatory Guidelines of April 2004 to detect abnormal specimens. The Proposed Guidelines of 2004 recommended the determination of immunoglobulin concentration of IgG on every oral fluid specimen. Concentrations of <0.1 ug/mL of IgG would indicate a substituted specimen. Some scientists believe that specimen validity testing is not needed for oral fluid specimens due to the observed collection process.

12. Confirmation Testing Chromatography + mass spectrometry Gas and liquid chromatography provide separation of analytes Detector is a mass spectrometer New confirmation technologies 1988-2010 only GC/MS permitted for urine confirmation drug testing New technologies permitted with Revised Guidelines (e.g. LC/MS, GC/MS/MS, LC/MS/MS) 2 NLCP labs currently using LC/MS/MS for 6-AM OF testing will require these new technologies Methodologies 12 January 27, 2011 The gold standard for confirmation drug testing is an analytical method that combines chromatographic separation and mass spectrometric identification. The two most common types of analytical chromatography methods used to separate compounds are gas and liquid chromatography. Either can then be interfaced with one or more mass selective detectors. GC/MS was the only confirmation method permitted for urine drug testing under the Mandatory Guidelines from 1988-October 2010. New technologies were permitted for urine drug confirmatory tests with the October 2010 Revised Guidelines. Some examples of the new technologies include LC/MS, GC/MS/MS, and LC/MS/MS. Of the 38 certified labs in the NLCP, only 2 labs are using a new technology. Both labs are using LC/MS/MS for 6-AM analysis which has the lowest urine confirmation cutoff and is subject to interference by other opioids. Oral fluid testing will require these new technologies.The gold standard for confirmation drug testing is an analytical method that combines chromatographic separation and mass spectrometric identification. The two most common types of analytical chromatography methods used to separate compounds are gas and liquid chromatography. Either can then be interfaced with one or more mass selective detectors. GC/MS was the only confirmation method permitted for urine drug testing under the Mandatory Guidelines from 1988-October 2010. New technologies were permitted for urine drug confirmatory tests with the October 2010 Revised Guidelines. Some examples of the new technologies include LC/MS, GC/MS/MS, and LC/MS/MS. Of the 38 certified labs in the NLCP, only 2 labs are using a new technology. Both labs are using LC/MS/MS for 6-AM analysis which has the lowest urine confirmation cutoff and is subject to interference by other opioids. Oral fluid testing will require these new technologies.

13. Confirmation Testing Confirmation test analytes and cutoff concentrations (ng/mL) Methodologies 13 January 27, 2011 This table displays the confirmatory test analytes and cutoff concentrations for oral fluid from the 2004 “Proposed Revisions to Mandatory Guidelines” compared to analytes and cutoffs for urine from the October 2010 “Revised Mandatory Guidelines”. Notice first that the analytes for oral fluid include two parent drugs not included in urine – THC parent and cocaine parent, and also includes the designer drugs MDMA, MDA and MDEA recently added to the urine panel. Also notice that the proposed confirmation cutoffs for oral fluid are lower than those for urine. The newer confirmation technologies will be helpful in the analysis of THC and will certainly be needed if the carboxy metabolite of THC now used in urine testing is added to the oral fluid panel since the concentration of this metabolite is usually in the picogram/mL concentration in oral fluid. This table displays the confirmatory test analytes and cutoff concentrations for oral fluid from the 2004 “Proposed Revisions to Mandatory Guidelines” compared to analytes and cutoffs for urine from the October 2010 “Revised Mandatory Guidelines”. Notice first that the analytes for oral fluid include two parent drugs not included in urine – THC parent and cocaine parent, and also includes the designer drugs MDMA, MDA and MDEA recently added to the urine panel. Also notice that the proposed confirmation cutoffs for oral fluid are lower than those for urine. The newer confirmation technologies will be helpful in the analysis of THC and will certainly be needed if the carboxy metabolite of THC now used in urine testing is added to the oral fluid panel since the concentration of this metabolite is usually in the picogram/mL concentration in oral fluid.