**3.3 Determining appropriate UDT cutoffs**

Sensitivity of detection currently used in many immunoassays may not be appropriate for the pain patient. This is because manufacturers set cutoffs for assays to identify overdose in emergency unit settings (Fraser & Zamecnik, 2003; Fraser, 2001; Hattab et al., 2000; Wingert, 1997). There is a need to establish appropriate cutoffs for patients on clinical doses of their medications rather than the high concentrations encountered in overdose situations. Specifically, studies have been conducted that better identify the appropriate cutoff for the pain patient population (Pesce et al., 2011).

One definition of appropriate cutoff levels is one that captures 97.5% or more of the population on a specific drug (Pesce et al., 2011). An example of the importance of setting appropriate cutoffs is for the drug clonazepam (West et al., 2010b). When measured by immunoassay using a nominal cutoff of 200 ng/mL, only 28% of the patients on the drug were determined to be compliant. When the same samples were measured by LC-MS/MS technique using a cutoff of 200 ng/mL, the group was found to be 70% compliant. Finally, when the LC-MS/MS cutoff was lowered to 40 ng/mL the group was 87% compliant. This study showed that first the immunoassay was insensitive in that the nominal 200 ng/mL cutoff did not apply to clonazepam, and second, a lower cutoff was needed to appropriately categorize compliance. Other studies have shown the need for lower cutoffs for pain medications (Mikel et al., 2009; Pesce et al., 2010a). As the consequences to the patient of dismissal from a practice can be very large and even life-changing (e.g., loss of insurance, loss of job or income), it is essential that physicians do not unjustifiably dismiss even a

Diagnostic Accuracy and Interpretation

**Drug**

**3.4 Confirmatory testing: mass spectrometry** 

laboratory error can be dismissed.

will describe later (Siuzdak, 2006).

site giving a positive test result.

**3.5 Test menu requirement** 

of Urine Drug Testing for Pain Patients: An Evidence-Based Approach 35

Cocaine 29.6 17 Marijuana 9.5 6.2 Methamphetamine 56.1 33.5

Table 5. Illicit Drug Cutoff Values. Modified with permission from West et al., 2011a.

Physicians dealing with pain patients not following the treatment plan or using illicit or non-prescribed medications, have difficulty with these situations (Jung & Reidenberg, 2007). The doctor must be absolutely confident that the test data from both the POC and laboratory conducting further testing is correct. By having positive results obtained in their offices as well as confirmatory laboratory data, physicians can confidently discuss expectations and behavioral changes with patients. Questions about laboratory mix-up of specimens or

Many laboratories performing UDT on the pain patient population typically test specimens by immunoassay and then follow this with confirmation by mass spectrometry (Cone et al., 2008). Mass spectrometry is an analytical technique that separates molecules based on their weight (mass) and fragmentation pattern. Identification is based on the fact that each drug has a specific mass and breakdown in the same way that each person has a specific fingerprint. A mass spectrometry instrument is usually coupled to a chromatographic column, in which the test drug, for example morphine, is separated from other components in the urine before submitting the sample into the mass spectrometer. The mass spectrometer identifies the test drug by its position in the chromatogram, the specific weight of the molecule, and by its fragmentation pattern. This technology is virtually foolproof. Mass spectrometry techniques are divided into two methods: gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Of the two, the newer LC-MS/MS is considered the gold standard, for reasons we

In cases where the physician wants the results immediately (within hours), confirmatory mass-spectrometry methods used at the most modern diagnostic laboratories provide results within 24-30 hours. As stated above, the major limitations of immunoassays are inappropriate cutoffs (sensitivity), varying specificity for individual drugs, and crossreactivity with other agents producing both false-negative and false-positive results (Manchikanti et al., 2008). The term cross reactivity is used to describe the reaction of an antibody with a chemical that is not the original immunizing drug. The reaction is poor because the affinity is much worse than the original drug. By poor we mean that at the same concentration of the original drug the test compound does not bind as well. However, as the concentration of the test compound is increased it eventually saturates the antibody binding

As mentioned earlier a broader clinical laboratory UDT menu is necessary to accurately monitor the pain patient population. Smaller hospitals as well as physician offices cannot

**Raw (ng/mL)** **CR Normalized (ng/mg CR)**

**Lower 2.5%**

single patient who is compliant with their medication regimens. This can be avoided by using appropriate cutoffs.

In an attempt to better define appropriate cutoffs for the pain patient population, the quantitative urine drug test results were examined for the prescription medications listed in Table 4. Using the criterion that the cutoffs should capture 97.5% of the examined population and employing the LC-MS/MS cutoffs listed in Table 4 showed it was possible to meet this standard (Pesce et al., 2011). One limitation of this approach is that the time after last dose and the dose itself were not known for these subjects. Regardless of the limitations of the study, the lower cutoffs provide results that can clearly identify compliance more accurately than other methods.


Table 4. Medication Cutoff Values. Modified with permission from Pesce et al., 2011.

As stated earlier, illicit drug use is common in this population (Madras et al., 2009; Schuckman et al., 2008). It stands to reason that identifying the appropriate illicit drug cutoffs for UDT is equally important. Using the same criterion as stated above, cutoffs for marijuana, cocaine, and methamphetamine have also been determined (Table 5) (West et al., 2011a). The lowering of these illicit drug cutoffs consistent with the latest SAMHSA guidelines in which the cocaine and amphetamine cutoffs were lowered to capture more illicit drug users (Federal Register, 2004).


Table 5. Illicit Drug Cutoff Values. Modified with permission from West et al., 2011a.

#### **3.4 Confirmatory testing: mass spectrometry**

34 Toxicity and Drug Testing

single patient who is compliant with their medication regimens. This can be avoided by

In an attempt to better define appropriate cutoffs for the pain patient population, the quantitative urine drug test results were examined for the prescription medications listed in Table 4. Using the criterion that the cutoffs should capture 97.5% of the examined population and employing the LC-MS/MS cutoffs listed in Table 4 showed it was possible to meet this standard (Pesce et al., 2011). One limitation of this approach is that the time after last dose and the dose itself were not known for these subjects. Regardless of the limitations of the study, the lower cutoffs provide results that can clearly identify

 7-Amino-Clonazepam 10 19 15 Alpha-Hydroxyalprazolam 10 15 11 Amphetamine 50 76 59 Buprenorphine 5 7 5 Carisoprodol 50 56 35 Codeine 25 29 15 Fentanyl 1 2 2 Hydrocodone 25 41 31 Hydromorphone 25 34 26 Lorazepam 20 30 25 Meperidine 25 88 28 Meprobamate 50 92 113 Methadone 50 89 74 Morphine 25 59 52 Oxycodone 25 45 46 Oxymorphone 25 44 38 Propoxyphene 50 60 42 Tapentadol 25 42 58 Tramadol 50 147 70

Table 4. Medication Cutoff Values. Modified with permission from Pesce et al., 2011.

As stated earlier, illicit drug use is common in this population (Madras et al., 2009; Schuckman et al., 2008). It stands to reason that identifying the appropriate illicit drug cutoffs for UDT is equally important. Using the same criterion as stated above, cutoffs for marijuana, cocaine, and methamphetamine have also been determined (Table 5) (West et al., 2011a). The lowering of these illicit drug cutoffs consistent with the latest SAMHSA guidelines in which the cocaine and amphetamine cutoffs were lowered to capture more

**Cutoff (ng/mL)**

**Estimated New Cutoff (Raw, ng/mL)** **CR Normalized Cutoff (µg/g creatinine)**

**Lower 2.5%**

using appropriate cutoffs.

compliance more accurately than other methods.

**Drug Analytical** 

illicit drug users (Federal Register, 2004).

Physicians dealing with pain patients not following the treatment plan or using illicit or non-prescribed medications, have difficulty with these situations (Jung & Reidenberg, 2007). The doctor must be absolutely confident that the test data from both the POC and laboratory conducting further testing is correct. By having positive results obtained in their offices as well as confirmatory laboratory data, physicians can confidently discuss expectations and behavioral changes with patients. Questions about laboratory mix-up of specimens or laboratory error can be dismissed.

Many laboratories performing UDT on the pain patient population typically test specimens by immunoassay and then follow this with confirmation by mass spectrometry (Cone et al., 2008). Mass spectrometry is an analytical technique that separates molecules based on their weight (mass) and fragmentation pattern. Identification is based on the fact that each drug has a specific mass and breakdown in the same way that each person has a specific fingerprint. A mass spectrometry instrument is usually coupled to a chromatographic column, in which the test drug, for example morphine, is separated from other components in the urine before submitting the sample into the mass spectrometer. The mass spectrometer identifies the test drug by its position in the chromatogram, the specific weight of the molecule, and by its fragmentation pattern. This technology is virtually foolproof. Mass spectrometry techniques are divided into two methods: gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Of the two, the newer LC-MS/MS is considered the gold standard, for reasons we will describe later (Siuzdak, 2006).

In cases where the physician wants the results immediately (within hours), confirmatory mass-spectrometry methods used at the most modern diagnostic laboratories provide results within 24-30 hours. As stated above, the major limitations of immunoassays are inappropriate cutoffs (sensitivity), varying specificity for individual drugs, and crossreactivity with other agents producing both false-negative and false-positive results (Manchikanti et al., 2008). The term cross reactivity is used to describe the reaction of an antibody with a chemical that is not the original immunizing drug. The reaction is poor because the affinity is much worse than the original drug. By poor we mean that at the same concentration of the original drug the test compound does not bind as well. However, as the concentration of the test compound is increased it eventually saturates the antibody binding site giving a positive test result.

#### **3.5 Test menu requirement**

As mentioned earlier a broader clinical laboratory UDT menu is necessary to accurately monitor the pain patient population. Smaller hospitals as well as physician offices cannot

Diagnostic Accuracy and Interpretation

**4. Interpretation of UDT results** 

of Urine Drug Testing for Pain Patients: An Evidence-Based Approach 37

The accurate interpretation of test results requires an understanding of the usefulness and limitations of immunoassays (Gourlay et al., 2010; Hammett-Stabler & Webster, 2008; Manchikanti et al., 2010; Nafziger & Bertino, 2009; Reisfield et al., 2007), a knowledge of opiate metabolism, and awareness of the expected ratios of the parent drug and its metabolites in urine (Reisfield et al., 2007). In addition, small amount of impurities in medications detectable by mass spectrometry can complicate the interpretation of UDT results. For example, codeine is present in morphine preparations and hydrocodone is present in oxycodone preparations (Evans et al., 2009; West et al., 2009, 2011b). Physicians who aren't aware of the presence of these impurities may wrongly dismiss a patient because he or she tested positive for codeine or hydrocodone when it was not prescribed. The presence of both parent drug and its metabolite in a urine sample readily measured by mass spectrometry can reassure the physician that the patient is taking the medication and that it is being metabolized appropriately. Also, for some drugs such as carisoprodol, fentanyl, or buprenorphine, only the metabolite may be observed. It is imperative that physicians prescribing these medications use a reference laboratory that is able to measure both the parent drug and its corresponding metabolite and be able to present interpretive results for the physician (Heltsley et al., 2010). Creatinine is a metabolic breakdown product that is present in urine. The amount of creatinine excreted into urine is nearly constant for any individual. Reference laboratories calculate the amount of drug excreted per gram of creatinine, which allows the monitoring of excreted medication or illicit drug over time. This information is useful to physicians in certain circumstances because some drugs, such as nordiazepam remain in the system long after a person stops taking them. A UDT result that is not corrected for creatinine may show that the patient is more positive for the drug than on a previous test, even though the patient has in fact stopped taking it. Except for changes in the patient's renal status, or loss from adipose tissue due to dieting, this conflicting result may be due to the second urine being more concentrated than the first. A creatinine-corrected value will correct for a patient's hydration on the day of the test and show a decrease in the amount of nordiazepam in the urine, thus supporting the patient's claim that he or she has stopped taking the drug. It is important that reference laboratories not only provide creatininecorrected results but that they give doctors or staff help in interpreting the data (Cone et al., 2009). It is also important for the physician to know if a patient has attempted to obscure UDT results by diluting a urine specimen. To accomplish this, he or she must have a grasp of creatinine and specific gravity UDT validity tests (Wu, 2001). Laboratory staff who

interface with clients should provide this information when questions arise.

As stated earlier, alcohol (ethanol) use among pain patients is a significant problem because of the risk for drug-drug interaction with opioid medication. For doctors to understand UDT ethanol results, it is essential that they understand ethanol metabolism and the formation of the ethanol byproducts ethyl glucuronide and ethyl sulfate (Crews et al., 2011a; Crews et al., 2011b; Dahl et al., 2002; Helander & Beck, 2005; Helander et al., 1996; Rosano & Lin, 2008; Schmitt et al., 1997; Stephanson et al., 2002; Wojcik & Hawthorne, 2007; Wurst et al., 2006; Wurst et al., 2004). This is because false positive ethanol results can result from fermentation of glucose from diabetic patient samples (Crews et al., 2011b). Crews et al. reported that about 1/3 of the ethanol positive samples were due to fermentation. Misinterpretation of

**5. Monitoring ethanol use in pain patients** 

meet this requirement. One reason for this is that immunoassays require separate analytical channels for each assay and this limits the number of tests a smaller laboratory may have in its menu (Olympus Au640 Product Information; Siemens V-Twin Analyzer Product Information; Thermo Fisher Mgc-240 Analyzer Product Information). Another reason is that certain drug tests may not exist for the laboratory's specific instruments, and the addition of another instrument is financially prohibitive, particularly if that instrument is a mass spectrometer (Agilent Technologies, Inc.). Many physicians treating the pain patient population send specimens to reference laboratories specifically designed to provide the required test menu to meet these needs. Tests for new drugs (i.e., tapentadol) (Nucynta - Tapentadol, 2010) or new illicit substances (i.e., K2, spice) (Sobolevsky et al., 2010; Vardakou et al., 2010) encountered in the pain patient population can be rapidly set up and validated on LC-MS/MS instrumentation. Therefore, this analytical technique is supplementing screening by immunoassay. Because of the limitations of immunoassays, confirmatory testing is essential for accurate clinical assessment of medication usage. With confirmatory testing, physicians have specific evidence of what medications a patient is or isn't taking. This assures the doctor that he or she is not discharging a patient inappropriately, and that care is appropriate and not limited.. The laboratories with the most advanced technology can eliminate the immunoassay step saving both the patient and the insurer money.

#### **3.6 Mass spectrometry as the gold standard for testing**

At this point in time, mass spectrometry is considered the method of choice for UDT analysis in pain management. This is because mass spectrometry offers the chromatographic separation and mass fragmentation patterns that are specific for the test medications such as opiates and benzodiazepines (Mohsin et al., 2007). In addition, this analytic approach uses isotope dilution to quantify the amount of drug in the urine specimen; isotope dilution is considered the gold standard for determining how much of a drug is in a specimen (quantitation) (Federal Register, 2004). This ability to quantify the amount of drug in urine has been proposed as a method of detecting drug abuse (Pesce et al., 2010c). However, it is important to note that it is not possible to relate the quantitative excretion of a drug to the drug dosage (Nafziger & Bertino, 2009). Quantitation of drugs using immunoassay technology is problematic, particularly if the antibody reagent cross reacts with multiple structurally related drugs; if the urine drug sample contains more than one drug in a class (i.e., hydrocodone and hydromorphone), the antibody reaction will vary with each drug present in the solution. This means that the assay cannot distinguish between the two drugs and give a reliable calculation of the amount of either drug present (Feldkamp, 2010).

Of the two commonly used mass spectrometry methods, LC-MS/MS offers several advantages over GC-MS (Mikel et al., 2010). These include the ability to discriminate a larger number of drugs in each test run, the very small amount of urine specimen required (as little as 25 microliters, or one drop), and the ability to use a sample that is neither derivatized nor extracted. This in turn has made possible the analysis of hundreds of urine specimens per day for a single mass spectrometer. Advances in the automated handling of specimens and bar coding allow for the accurate processing of thousands of samples per day. This method of analysis can provide physicians with results more rapidly than by GC-MS (Mikel et al., 2010).
