Current methodologies for drugs of abuse urine testing, part 1

Abstract

Analysis of drug panels in urine samples can be challenging, and the trend towards larger panels including multiple drug classes compounds the issues faced during method development. This white paper examines a number of aspects of sample preparation, and their impact on the success of subsequent LC-MS/MS analysis of broad urine panels.
This examines the applicability of various sample preparation techniques: supported liquid extraction, reverse phase SPE and mixed-mode SPE, to the various classes of drugs extracted. In addition, hydrolysis approaches: enzyme type and protocol used (time, temperature), are compared. Mixed-mode (reverse phase/cation exchange) SPE is widely used for extraction of basic drug classes from urine, but the inclusion of drugs and metabolites that exhibit ‘non-typical’ functionality within urine panels can be problematic.

Introduction: Practical considerations for LC/MS method development of a comprehensive urine pain panel

Urine drug testing to support pain management is a mainstay of the clinical toxicology laboratory. Reduced reimbursement has continued to put increasing pressure on laboratories. Many toxicology labs are moving to larger drug panels to increase throughput and efficiency, while reducing turnaround time and cost. LC-MS methods with 50 or more drugs and metabolites are common. While “dilute and shoot” (D&S) methods are easy and affordable, they can result in shortened LC column lifetimes and increased MS instrumentation downtime. Matrix effects can also affect sensitivity and overall method performance.

Clean-up of hydrolysed urine specimens can reduce matrix effects, increase LC column lifetimes, and keep MS instrumentation cleaner. The result is an overall increase in efficiency and productivity because of reduced downtime. Samples can also be concentrated, resulting in improved sensitivity.

Here, we present sample preparation considerations for a panel of 56* drug analytes by three different sample preparation methods: supported liquid extraction (ISOLUTE® SLE+), polymeric reverse phase solid phase extraction SPE (EVOLUTE® EXPRESS ABN), and mixed-mode polymeric reverse phase strong cation exchange SPE (EVOLUTE® EXPRESS CX). Details of the sample preparation methods used are shown below.

Importance and role of logP and pH

The selection of sample preparation products and protocols should be determined based on the chemical properties of the compounds of interest. These properties determine how the compounds are retained and eluted using different sample preparation methods. The octanol-water partition coefficient (logP) is a measure of the hydrophobicity of an analyte. It is an indication of an analyte’s reverse phase retention behaviour and its ability to partition into an organic solvent when using supported liquid extraction (SLE). The higher the logP, the more hydrophobic the compound. The acid dissociation constant (pKa) is the pH where a compound is 50% ionized and 50% non-ionized. Figure 1 demonstrates behaviour of an acidic and basic compound under different pH conditions. Pretreatment of specimens should be ±2 pH units away from the pKa to make sure a compound is completely ionized or neutral, depending upon the mechanism of retention of the sample preparation product.

Acid Analyte Plot (top) Base Analyte Plot (bottom)

Figure 1. Ionization at different pH conditions for an acidic and basic analyte with a pKa of 8.0.

ISOLUTE® SLE+ for sample preparation

Supported liquid extraction (SLE)

The mechanism for SLE is similar to liquid-liquid extraction, but instead of partitioning between an aqueous and organic phase in a tube or vial, aqueous samples are absorbed onto a refined diatomaceous earth based sorbent (see Figure 2). The aqueous sample is loaded onto the ISOLUTE® SLE+ column and dispersed as small droplets. A water-immiscible organic solvent, typically ethyl acetate (EtOAc), dichloromethane (DCM) or methyl tert-butyl ether (MTBE) is used to elute the analytes of interest. Compounds
of interest partition into the elution solvent and are collected. The addition of a polar modifier such as 2-propanol (IPA) can aid in the elution of more hydrophilic compounds. Interfering or undesirable water-soluble compounds are retained on the ISOLUTE SLE+ column, providing a clean extract. This extraction method works well for acidic, basic and neutral compounds, and is based on the logP of the compound.

Figure 2. Mechanism for supported liquid extraction (SLE) using ISOLUTE® SLE+ products. Diagram illustrates a column or individual well of 96-well plate.

EVOLUTE® EXPRESS for sample preparation

Solid-phase extraction (SPE)

SPE methods depend upon retention of the analytes of interest, removal of interferences by washing with aqueous and/or organic solvents, followed by elution of the targeted drugs and metabolites (see Figure 3). Retention and elution are based on normal, reverse phase, or ion exchange mechanisms. EVOLUTE® EXPRESS ABN is a polymeric reverse phase SPE sorbent. EVOLUTE® EXPRESS CX is a mixed-mode polymeric strong cation exchange product, which can exhibit both reverse phase and ion exchange retention behaviour. Sample pretreatment and wash steps must be carefully controlled and understood. The retention mechanism of the compounds of interest must be known to ensure that analytes are not lost during the load step, washed away during interference wash steps, or retained on the column during elution, especially when compounds are retained and eluted by both mechanisms.

With a large panel of 50 or more drugs and metabolites, it will be very difficult to find conditions where all compounds are in the same ionization state. The method must be able to accommodate acidic, basic and neutral compounds in a single sample preparation method.

Figure 3. General SPE method procedure

Properties of analytes

The compounds evaluated for this section and their logP and pKa values are listed in Table 1. The logP and pKa were sourced from chemicalize.com or the Human Metabolome Database (1–2).

Experiments in hydrolysis

Urine hydrolysis considerations

Most drugs are metabolized prior to excretion in the urine or faeces. Many drugs and metabolites are conjugated as a glucuronide to increase water solubility and improve
elimination from the body. Hydrolysis of urine specimens using a beta-glucuronidase enzyme to convert the metabolites to their “free” form for analysis increases assay sensitivity.

Three different sample preparation techniques for extraction of free drugs from hydrolysed urine were investigated for this study. In addition, we evaluated four different
beta-glucuronidase enzymes with eight different glucuronide compounds over several incubation times and temperatures to determine optimal hydrolysis conditions for selected drug classes.

Sample preparation methods

For EVOLUTE® EXPRESS CX methods, 100 µL of urine spiked at 100 ng/mL with each drug was combined with 100 µL of 0.1M acetate buffer, pH 4.0, 0.15M sodium phosphate buffer, pH 6.8, or 25 µL IMCS buffer, depending on the enzyme used. Enzyme was added. Various hydrolysis conditions were evaluated (see Hydrolysis Evaluation Procedure). Following hydrolysis, 100 µL of 4% phosphoric acid (H3PO4) was added to all samples before proceeding with the extraction.

For ISOLUTE® SLE+ and EVOLUTE EXPRESS ABN methods, 100 µL of urine spiked at 100 ng/mL with each drug was combined with 100 µL of 0.1M acetate buffer, pH 4.0, 0.15M sodium phosphate buffer, pH 6.8, or 25 μL IMCS buffer, depending on the enzyme used. Enzyme was added. Various hydrolysis conditions were evaluated (see Hydrolysis Evaluation Procedure). Following hydrolysis, 100 μL of 0.1% NH4OH was added to all samples before proceeding with the extraction.

The extraction methods used are detailed below. Samples were loaded onto one of three different extraction columns (all 96-well plate format):

• ISOLUTE® SLE+ 400 µL 96-well plate, (820-0400-P01)
• EVOLUTE® EXPRESS ABN 30 mg plate (600-0030-PX01)
• EVOLUTE® EXPRESS CX 30 mg plate (601-0030-PX01)

 

Hydrolysis evaluation procedure

Three beta-glucuronidase enzymes were evaluated: red abalone (BG100, Kura Biotec, Los Angeles, CA), abalone (Campbell Science, Rockford, IL), along with a recombinant enzyme (IMCSzyme, Irmo, SC). An additional recombinant enzyme was added for later studies (BGTurbo, Kura Biotec, Los Angeles, CA). The enzymes were evaluated to determine which provided the most complete hydrolysis of glucuronide metabolites without affecting the overall recovery of non-conjugated compounds.

Four glucuronides were included in a urine glucuronide control to determine the extent of hydrolysis by each enzyme: morphine-3-beta-D-glucuronide, norbuprenorphine
glucuronide, oxazepam glucuronide, and 11-nor-9-carboxy-THC glucuronide (THC-COOH) (Cerilliant, Round Rock, TX). Four additional controls were added to later studies: codeine-3-beta- D-glucuronide, hydromorphone glucuronide, oxymorphone glucuronide, and lorazepam glucuronide, (Cerilliant, Round Rock, TX). The control was prepared so that the amount of non-conjugated drug would equal 100 ng/mL upon hydrolysis.

A spiked urine sample containing 56 non-conjugated drugs and metabolites at 100 ng/mL was also analysed to calculate hydrolysis efficiency and compare differences in matrix effects among the 3 or 4 enzymes and hydrolysis conditions. Sample volume for all enzymes was 100 µL. For the Campbell and BG100 enzymes, 100 µL of 0.1M ammonium acetate buffer, pH 4.0 was added. Next, 20 µL of enzyme was added. For the IMCSzyme, 25 µL of IMCS buffer and 55 µL of water was added to the sample. Next, 20 µL of enzyme was added. For the BGTurbo enzyme, 100 µL of 0.15M sodium phosphate buffer, pH 6.8 and 55 µL of water was added to each sample. Next, 30 µL of enzyme was added. For analysis of the Campbell, BG100 and IMCS enzymes, samples were incubated at either 55 °C or 65 °C for 30 or 60 minutes. All enzymes, including the BGTurbo enzyme, were also analysed at room temperature for 30 minutes and at 55 °C for 10 minutes. The samples were extracted using EVOLUTE® EXPRESS CX method described below.

ISOLUTE® SLE+ method

EVOLUTE® EXPRESS ABN method

EVOLUTE® EXPRESS CX method

Results

Urine hydrolysis results

For the 65°C hydrolysis, the results indicate that no one enzyme performed better for the four initial glucuronides tested. The 55oC 60 minute incubation did not show improved hydrolysis efficiency for most enzymes or glucuronides (see Figure 4). When adding in the BGTurbo enzyme for comparison, as well as additional glucuronide compounds, the room temperature incubation was not sufficient for hydrolysis, except in the case of lorazepam and oxazepam (see figure 5). The 55 °C, 10 minute incubation showed similar results, in that lorazepam and oxazepam were close to fully hydrolysed while other glucuronides, particularly opiates, were not well hydrolysed (see figure 6). When hydrolysing at 55 °C for
30 minutes, close to complete hydrolysis was observed for most glucuronidase compounds (see figure 7). Therefore, this 55 °C, 30 minute incubation was used for all extraction techniques.

Hydrolysis of THC-COOH was temperature and time dependent for the enzymes tested (the degree of hydrolysis was higher at lower times and temperatures). Recovery of most of the analytes in the 56 compound “free” control was consistent (within ±10%) among the three enzymes at the various times and temperatures. Carisoprodol, hydromorphone, and zolpidem-phenyl-4-COOH showed some variability among different enzymes and incubation parameters. Figure 8 shows the recoveries for these compounds for three enzymes (Campbell, BG100, and IMCSzyme) under all hydrolysis conditions.

Based on these results, the Campbell enzyme provided adequate hydrolysis efficiencies for most of the glucuronide compounds when a 30 min, heated incubation was used. The BG100, BGTurbo, and IMCS enzymes provided more complete hydrolysis efficiency of the opiate compounds. However, a heated incubation of at least 30 minutes is needed to achieve adequate hydrolysis.


Figure 4. Percent hydrolysis (calculated as the ratio of glucuronide/free) for each of the compounds in the glucuronide control for three enzymes and hydrolysis conditions.

Figure 5. Percent Hydrolysis (calculated as the ratio of glucuronide/free) at room temperature for 30 minutes. (Figure includes data for additional glucuronide analytes and BGTurbo enzyme).

Figure 6. Percent Hydrolysis (calculated as the ratio of glucuronide/free) at 55 oC for
10 minutes. (Figure includes data for additional glucuronide analytes and BGTurbo enzyme).
Figure 7. Percent Hydrolysis (calculated as the ratio of glucuronide/free) at 55 oC for
30 minutes. (Figure includes data for additional glucuronide analytes and BGTurbo enzyme).

Figure 8. Recoveries of carisoprodol, hydromorphone and zolpidem-phenyl-4-COOH for three enzymes and all hydrolysis conditions.

Results by drug class

Opiates/Opioids I

Codeine, morphine, hydrocodone, hydromorphone, oxycodone, oxymorphone, 6-monoacetylmorphine (6-AM) and metabolites.

Greater than 90% recovery was achieved using EVOLUTE® EXPRESS CX with acid pretreatment, 50% MeOH wash, and eluting with 78:20:2 (v/v) DCM:IPA:NH4OH. ISOLUTE® SLE+ provided >90% recovery for all compounds except 85% recovery for n-desmethyltapentadol and 70% recovery for norhydrocodone and morphine using elution with 95:5 (v/v) DCM:IPA. EVOLUTE® EXPRESS ABN yielded 30–60% recovery for 6-AM, hydrocodone, norhydrocodone, codeine, dihydrocodeine, oxycodone and <10% recovery for morphine, hydromorphone, oxymorphone, using basic pretreatment, 10% MeOH wash, and eluting with 90:10 (v/v) DCM:IPA.

Opioids and opioid agonists

Methadone, buprenorphine, fentanyl, meperidine, tramadol, tapentadol, naloxone and metabolites.

Better than 80% recovery of all compounds was observed using the described protocol with 78:20:2 (v/v) DCM:IPA:NH4OH elution and EVOLUTE® EXPRESS CX. ISOLUTE® SLE+ produced >90% recovery for all compounds except for norfentanyl and n-desmethyltapentadol (80% recovery) with a 95:5 (v/v) DCM:IPA elution. EVOLUTE® EXPRESS ABN showed higher recovery for this group of compounds than the opiates/ opioids I group with >80% recovery for all compounds except for 50–60% recovery for norbuprenorphine, norfentanyl, tapentadol and n-desmethyltapentadol, and 25% recovery for naloxone. Basic pretreatment and 10% MeOH wash with an elution with 90:10 (v/v) DCM:IPA were used.

Benzodiazepines and Z-drugs

Alprazolam, chlordiazepoxide, clonazepam, diazepam, lorazepam, midazolam, zolpidem and metabolites

90% recovery or better was attained using EVOLUTE® EXPRESS CX and ISOLUTE® SLE+, using previously described protocols, except for recovery of 7-aminoclonazepam (80%) and the recovery of zolpidem-phenyl-4-COOH (80% when using CX and 50% when using SLE+). The elution solvent for EVOLUTE® EXPRESS CX was 78:20:2 (v/v) DCM:IPA:NH4OH. EVOLUTE® EXPRESS ABN gave recoveries of >80% recovery
for all compounds except for 7-aminoclonazepam, which had a recovery of 50%.

Barbiturates

Butalbital, pentobarbital, phenobarbital, secobarbital

EVOLUTE® EXPRESS CX produced poor recovery for all barbiturates under the conditions evaluated. These drugs are weakly acidic and would not be expected to perform well on a cation exchange SPE phase and with acid pretreatment. Recovery of >90% was achieved using ISOLUTE® SLE+ and EVOLUTE® EXPRESS ABN.
 

Stimulants

Amphetamine, methamphetamine, ritalinic acid, MDMA, cocaine, benzoylecgonine (BZE)
EVOLUTE® EXPRESS CX had recoveries of >90% for all compounds except ritalinic acid which had 30% recovery using acid pretreatment, 50% MeOH wash and elution with 78:20:2 (v/v) DCM:IPA:NH4OH. Recovery for ritalinic acid was increased to 80% by changing the elution solvent to 78:20:2 (v/v) DCM:MeOH:NH4OH. These samples were not as clean as samples eluted with DCM:IPA:NH4OH. ISOLUTE® SLE+ provided recoveries of >80% for methamphetamine, MDMA, cocaine and BZE, 50% recovery of amphetamine and <10% ritalinic acid.

EVOLUTE® EXPRESS ABN had >80% recovery for cocaine and BZE, <30% recovery for amphetamine and methamphetamine, and 50% recovery of ritalinic acid. No other elution solvents or pretreatments were evaluated.

Illicit drugs and tricyclic antidepressants (TCAs)

11-nor-9-carboxy-delta-9-tetrahydrocannabinol (9-carboxy-THC), phencyclidine (PCP), ketamine, norketamine, amitriptyline, nortriptyline

EVOLUTE® EXPRESS CX and ISOLUTE® SLE+ yielded recoveries of >90% for all analytes except 9-carboxy-THC (80%). EVOLUTE® EXPRESS ABN produced >80% recovery of ketamine, norketamine, and PCP, 70% recovery for 9-carboxy-THC, and approximately 70% recovery for amitriptyline and nortriptyline.

Anticonvulsants, carbamates

Pregabalin, gabapentin, meprobamate, carisoprodol

The recovery of these compounds is difficult in a large drug panel. Extensive work was conducted to find the best conditions for recovery of these analytes, and is the subject of section 2 of this white paper. Less than 20% recovery of pregabalin, gabapentin, carisoprodol and meprobamate was observed using EVOLUTE® EXPRESS CX with acid pretreatment, 50% MeOH wash and elution with 78:20:2 (v/v) DCM:IPA:NH4OH. Lowering the organic wash to 30% MeOH improved recovery of meprobamate. Replacing IPA in the elution solvent with MeOH improved recovery to 80% for pregabalin, 100% for gabapentin and 30% for carisoprodol, but samples are not as clean as those eluted with IPA. ISOLUTE® SLE+ gave >90% recovery of carisoprodol and meprobamate, but <20% recovery of pregabalin and gabapentin. EVOLUTE® EXPRESS ABN provided >80% recovery of carisoprodol and meprobamate and <20% recovery of pregabalin and gabapentin.

Removal of conditioning and equilibrating for EVOLUTE® EXPRESS ABN and EVOLUTE® EXPRESS CX

ABN and CX are polymeric sorbents with hydrophilic (water wettable) characteristics. Because of this, the conditioning and equilibrating steps can be removed. This provides a reduction in both cost and volume of solvents used. Figure 9. shows a comparison of several compounds with and without conditioning and equilibrating for the IMCSzyme when extracting using ABN. As can be seen, there is little difference between conditioning and no conditioning. The no conditioning extraction tends to have slightly higher recoveries than the conditioning extraction. Figure 10. shows recoveries for several compounds extracted using CX with and without conditioning and equilibrating. Similar to ABN, the recoveries are very similar between the two extractions.
 
Figure 9. Selected analyte recoveries with and without conditioning and equilibrating steps using the IMCSzyme enzyme for hydrolysis, on EVOLUTE® EXPRESS ABN.

Figure 10. Selected analyte recoveries with and without conditioning and equilibrating steps using the IMCSzyme enzyme for hydrolysis, on EVOLUTE® EXPRESS CX.

General recommendations

We have presented three approaches for extraction and clean-up of 56 drugs and metabolites. The choice of the final method depends on the drugs of interest and their properties. ISOLUTE® SLE+ is recommended if opiates, opioids, benzodiazepines, stimulants (except ritalinic acid), PCP, barbiturates, 9-carboxy-THC, TCAs, meprobamate,
carisoprodol, ketamine and norketamine are in the panel.

If the drugs and metabolites in the urine panel are mostly basic: opiates, opioids, benzodiazepines, PCP, stimulants (except ritalinic acid), TCAs, ketamine, norketamine, and 9-carboxy-THC; then the EVOLUTE® EXPRESS CX method with sample pretreatment using 4% H3PO4, a 50% MeOH wash, and elution with 78:20:2 (v/v) DCM:IPA:NH4OH is recommended. If ritalinic acid, gabapentin and pregabalin are required, the same protocol, except elution with 78:20:2 (v/v) DCM:MeOH:NH4OH should be used.

EVOLUTE® EXPRESS ABN works well for some opioid drugs and metabolites, most benzodiazepines, ketamine, norketamine, PCP, 9-carboxy-THC, amitriptyline, nortriptyline, carisoprodol, meprobamate, cocaine and BZE. The organic wash must be limited to 10% MeOH because of the reverse phase retention mechanism. Amphetamine, methamphetamine and ritalinic acid had lower recoveries (30–50%) but this could be adequate depending upon the sensitivity required.

Lastly, the conditioning and equilibrating steps are not necessary to achieve maximum recoveries when extracting using EVOLUTE® EXPRESS ABN or EVOLUTE® EXPRESS CX. The
elimination of those steps can save time and money. Developing a single sample preparation method for a large panel of drugs and metabolites can be challenging. Finding a single sample preparation protocol for 50+ compounds requires knowledge of the hydrophobicity and acid-base properties of the drug analytes. Compromises in recovery and sample cleanliness are inevitable when multiple drug classes with vastly different properties are required in a single method. Methods should be optimized to provide sufficient recovery for required sensitivity and sample cleanliness. Several approaches should be investigated and evaluated to provide the most rugged, robust and sensitive method. Urine specimens should undergo enzymatic hydrolysis to maximize recovery of drug analytes that are conjugated prior to elimination in the urine. The enzyme and conditions for both hydrolysis of glucuronide metabolites and recovery of non-conjugated compounds should be selected based on the compounds of interest and the required limits of detection.

References

1. Chemicalize.com, http://www.chemicalize.com, accessed August 1–10, 2016.
2. Human Metabolome Database, http://www.hmdb.ca/ metabolites, accessed August 1–10, 2016.
   
   

Literature number: PPS443 (part 1)