R eview For reprint orders, please contact [email protected]

The challenges of LC–MS/MS ana­lysis of opiates and opioids in urine Opioids are some of the most commonly prescribed and abused drugs around the world. Primarily used for a­nesthesia or pain management, other opioids can also be used in the treatment of opioid addiction. Given these facts, clinicians often randomly test or monitor their patients to determine compliance or abstinence from these drugs via immunoassay methods. When a positive screen is obtained, a confirmatory assay is carried out and ­although the gold standard has been GC–MS, LC–MS/MS is fast becoming a valid and popular alternative. This review will discuss opioids, the complex metabolic pathways, the measurement of these drugs, the challenges i­nvolved and, finally, will describe some LC–MS/MS methods published from 2003 until 2013. What are opiates & opioids? Opiates are naturally occurring products derived from the opium poppy, Papaver somniferum, and include morphine, codeine, thebaine, papa­verine and noscapine. They have analgesic properties [1] and exert their effects by binding to and activating G-protein-coupled opioid receptors (m, d and k) in the CNS, peripheral tissues and the reproductive tract where they cause a reduction in pain, sedation, euphoria and respiratory depression, and in the gastrointestinal tract where they reduce gut motility [2–4]. The term opioid is essentially a broad term covering all compounds with morphine-like action, although they may have distinct chemical structures. These include endogenous opioids, of which at least ten have been identified in the brain belonging to distinct families (endorphins, enkephalins, dynorphins, nociceptin/orphanin FQ, endmorphins and morphiceptin)[5], and the so-called semi-synthetic or synthetic opioids, which include diacetylmorphine (h­eroin), hydromorphone, hydrocodone, oxycodone, oxymorphone, fentanyl, methadone, buprenorphine and naloxone. Use & abuse of opioids Opioids are most commonly clinically used for anesthesia or pain management purposes and in the case of buprenorphine and methadone may also be used for opioid-replacement therapy in patients with opioid dependence. Naloxone is a competitive m-opioid receptor antagonist and is used to reverse the life-threatening respiratory system depression that occurs in opioid overdose, and it is also a component of Su­boxone®, a treatment used for opioid dependence [6]. The

recognition of chronic pain as a significant problem in the last 20 years has led to a dramatic increase in the number of opioids prescribed for pain management purposes. In 2011, hydro­ codone was the most prescribed drug in the USA, and in fact in recent years, Americans consumed 99% of the hydrocodone used worldwide, even though they make up less than 5% of the world’s population [7,8]. Analgesic drugs were the substance class most frequently involved in human toxic exposures (11.7%) in the USA in 2011, and within this drug class, opioids were involved to some extent in 8.8% of all poisonrelated fatalities reported to the American Association of Poison Control Centers [9]. Part of the issue is that opioids are also commonly abused or used illicitly either by taking or being given someone else’s prescription medication, by falsely obtaining a prescription from a doctor or, in only a small percentage of cases, by buying the drugs from drug dealers. Excessive use of opioids can lead to tolerance and addiction through drug-induced neuronal dysfunction and neurotoxicity. This is due to the fact that these drugs imitate the endogenous opioid neuropeptides that activate the same receptors resulting in dopamine release, which has been associated with reward mechanisms [10]. Opioid addiction is associated with a poor quality of life, and with comorbid psychiatric symptoms and personality disorders [11]. Behavioral patterns of addiction are complex with the main characteristic being compulsive drug use despite the negative consequences, such as a decrease in social functioning, deterioration of health, deficits in cognitive function and motivation, and the high probability of drug re-use even after

10.4155/BIO.13.244 © 2013 Future Science Ltd

Bioanalysis (2013) 5(22), 2803–2820

Deborah French University of California San Francisco, 185 Berry Street, Suite 290, San Francisco, CA 94107, USA Tel.:+1 415 514 6627 Fax:+1 415 353 1178 E-mail: [email protected]

ISSN 1757-6180

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French prolonged drug-free periods [12]. Treatment for opioid abuse and addiction is extraordinarily expensive in terms of workplace costs such as lost productivity, healthcare costs and criminal justice costs [13]. Opioid drugs, therefore, have a significant socioeconomic impact and are a significant cause of morbidity and mortality in the USA and around the world.

Key Terms Opioid receptors: Proteins in the cell membrane that endogenous or exogenous opioids bind to and activate in order to exert their effects.

Opioid: A compound that has morphine-like action in that it acts on the opioid receptors although it may be structurally distinct from morphine itself.

if heroin was administered almost immediately before the bladder is voided, although there are reports of very small quantities of these compounds in urine 40 h after heroin administration [15]. The presence of 6MAM in the urine is therefore a very specific marker of heroin use [14]. Another heroin metabolite, 6-acetylcodeine, is a by-product of illicit heroin synthesis and is often found in the urine of heroin users. Morphine is a metabolite of heroin, but it is also a prescription drug in its own right. The major metabolic pathway of morphine involves the conjugation of a glucuronide group by UGT2B7 to form morphine-3b-glucuronide (~75% of excreted morphine; inactive) and morphine-6b-glucuronide (active) [15–19]. Additionally, morphine can be N-demethylated by cytochrome P450 (CYP)3A4 and to a lesser extent CYP2C8, to form normorphine [20,21]. Codeine can also be formed in the metabolism of heroin, but is also a prescription drug. Codeine is metabolized by O-demethylation to morphine [22], a reaction that

Metabolism of opioids The metabolism of opiates and opioids is somewhat complex, but should be understood by the clinicians prescribing these drugs and ordering the urine drug testing and by the clinical laboratory in which the testing is performed (Figure 1). After administration, heroin is rapidly deacetyl­ ated in the blood to 6-monoacetylmorphine (6MAM), and 6MAM is then quickly deacetyl­ ated to morphine. As the half-lives of heroin and 6MAM are approximately 5 and 45 min, respectively [14], detection of these compounds in a urine drug screen will usually only occur

Opioid dependence:

Physically dependent on opioid-like drugs illustrated by development of tolerance to these drugs such that larger quantities of the drugs are required to produce the same analgesic effects (desensitization of the opioid receptors) and withdrawal symptoms (agitation, anxiety and insomnia) should the drugs not be taken.

Methadone

Heroin

1

16 6-monoacetylmorphine

6-acetylcodeine

EDDP

2 5

Nomorphine

3

Morphine

Codeine

4 Morphine-3- and -6-glucuronide

6

EMDP

7 8

Hydromorphone

Hydrocodone

10

9

Codeine-6glucuronide

Norhydrocodone

Hydromorphone3-glucuronide

Buprenorphine

Oxymorphone

Oxymorphone-3glucuronide

11 Noroxycodone

14

18

Norbuprenorphine

19

Oxycodone

12

15

17

Norcodeine

Buprenorphine-3glucuronide

20 Norbuprenorphine -3-glucuronide

13

Noroxymorphone

Naloxone 22

Found in illicit preparations of heroin

Opiate

Minor metabolic pathway

Opioid

Naloxone-3glucuronide

Fentanyl 21 Norfentanyl

Figure 1. Metabolism of selected opiates and opioids. (1) Carboxylesterase; (2) carboxylesterase; (3) CYP2D6; (4) UGT2B7; (5) CYP3A4/CYP2C8; (6) CYP3A4; (7) UGT2B7; (8) CYP2D6; (9) CYP3A4; (10) UGT2B7; (11) CYP3A4/5; (12) CYP2D6; (13) CYP3A4; (14) CYP3A4; (15) UGT2B7; (16) CYP3A4, 2C19, 2B6 and 2D6; (17) CYP3A4; (18) CYP3A4 and 2C8; (19) UGT1A1; (20) UGT2B7 and 1A3; (21) CYP3A4 and (22) UGT2B7.

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future science group

| R eview

The challenges of LC–MS/MS ana­lysis of opiates and opioids in urine is catalyzed by CYP2D6, and it is also glucuronidated to the active codeine-6b-glucuronide by UGT2B7 [23,24]. Similar to morphine, codeine can also be N-demethylated to form norcodeine [25]. Minor metabolic pathways for morphine and codeine are the formation of hydromorphone and hydrocodone, respectively, which are opioid drugs commonly prescribed by clinicians [26,27]. Hydrocodone is converted by O-demethylation to hydromorphone by CYP2D6, and by N-demethylation to nor­hydrocodone by CYP3A4 [28–30]. Hydromorphone is glucuronidated by UGT2B7 to hydromorphone‑3b-glucuronide, an active metabolite [18]. Oxycodone is metabolized to oxymorphone by O-demethylation via CYP2D6 and predominantly to noroxycodone by N-demethylation via CYP3A4/5 [31–33]. Noroxycodone undergoes further O-demethylation to produce noroxymorphone, which is also produced from the N-demethylation of oxymorphone as a H3C

O

minor pathway [33]. Oxymorphone can also be conjugated with a glucuronide group to form ­oxymorphone-3b-glucuronide [34]. Fentanyl undergoes N-dealkylation to norfentanyl via CYP3A4 [35]. Methadone is N-demethyl­ ated via CYP3A4, 2C19, 2B6 and 2D6 to form EDDP which is subsequently N-demethylated via CYP3A4 to form EDMP [36]. Buprenorphine is N-dealkylated via CYP3A4 and 2C8 to norbuprenorphine and both of these compounds are glucuronidated by UGT1A1, and by UGT2B7 and UGT1A3, respectively [36]. Finally, naloxone is glucuronidated by UGT2B7 to form naloxone-3-glucuronide [18].

Key Terms Illicit: The illegal use of opioid drugs.

Glucuronidated: During

Phase II metabolism of opioid drugs, a glucuronic acid group is added enzymatically to specific compounds to make them more water soluble to aid in the excretion of these drugs via the kidneys.

Accurate quantification of opioids is necessary Performing urine toxicology testing is an integral part of caring for patients in addiction or pain management settings [37–40]. The major reasons for monitoring opioid use in patients HO

O

HO

O

H3C

O O

H

O H

O N

O

H3C

O

H

O H

CH3

O

N CH 3

O

H3C

HO

NH

O

H

H

OH

O

HO

Norcodeine

H

HO

H

OH

N

O

H

N CH 3

O Hydromorphone

Morphine-3-glulcuronide

Codeine-6-glulcuronide

Hydrocodone

OH

CH3

HO

OH O

H3C

H

OH

O

O

HO OH

N

N CH 3

H O

OH

HO

H

H

HO

OH

H

HO N

H3CO

O

N H O

Morphine-6-glulcuronide

H

O

O

OH

HO O

O

Codeine

OH OH

O

H

O

Normorphine

HO

HO

O

O H

N CH 3

HO O

H

N CH 3

Morphine

H

O

H

H

HO

6-acetylcodeline

N H3CO

O

H

H

N O

6-monoacetylmorphine

Heroin

O

H

O

O

H O

H

Hydromorphone-3-glucuronide

O

O NH

O O

Norhydrocodone

Figure 2. Selected opiates and opioids.

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French are to determine if a patient prescribed opioids is achieving therapeutic concentrations or if a dose adjustment is required; to determine if a patient is compliant with their medication; to determine if the patient is diverting their prescribed medication, in other words, if they are not taking their prescribed opioids and, for example, are selling them to other people; and finally, to determine that patients are not taking any opioids illicitly. Therefore, it is obviously important that false-negative or false-positive results be avoided, as should indeterminate results that could be caused by analytical i­nterferences [41,42]. The standard of care is to perform a urine drug screen, predominantly carried out by immunoassay methodologies. If the screen for a particular class of drugs is positive, confirmatory testing can be carried out. This confirmatory testing should be more specific than the screen in that it should be able to detect specific drugs, not just drug classes, to help clinicians determine what opioids have or have not been taken based upon the complex metabolic pathway. This is commonly accomplished by MS coupled with GC or LC [43]. Urine is the biological matrix of choice for this type of testing as its collection is much less invasive and inconvenient than, for example, blood, and additionally, opioids can be detected for longer and at higher concentrations than in other matrices. A disadvantage of urine testing is that it is more easily manipulated or adulterated than other matrices; however, testing for creatinine and checking the specific gravity of the urine can help authenticate that it is a genuine urine sample [44,45].

Key Terms Isomers: Compounds that

have the same chemical formula, but with different structural formulas (e.g., morphine and hydromorphone).

Parent drug: The form of the

drug that is initially consumed before it has been metabolized or the fraction of drug excreted unchanged in the urine.

Matrix effects: Components

of the biological sample (e.g., urine, plasma or serum) elute from the chromatography column at the same time as the analyte to be quantified producing either a decrease or an increase in the ionization efficiency of that analyte in the mass spectrometer.

HO

O

O

OH

O N

O

N

O Oxymorphone

HOOC HO HO

OH

H

Oxycodone NH

O

HO

O

H

OH O H

O

OH N CH3

O Oxymorphone-3-glucuronide

OH

OH NH

O

H3CO

Noroxymorphone

O

Noroxycodone

Figure 3. Oxycodone and its metabolites.

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Challenges of opioid measurement Detection of opioids and their metabolites is particularly challenging due to the structural similarities and isomeric nature of these compounds (Figures 2–5). MS lends more specificity to quantifying opioids than antibody-based immunoassay methodologies [41,43]. However, if the opioids are indeed isomers, mass spectrometers cannot distinguish between them and, therefore, the specificity must be achieved in the LC method by separating these isomers in time on the chromatography column before they enter the mass spectrometer [46]. Additionally, as discussed above, depending on the opioid used, the detection window for the parent drug in urine could be in the order of minutes or hours, and therefore it is important to be able to detect the metabolites of these compounds in order to aid identification of which opioid was administered, and to ensure that a positive result is indeed obtained in patients prescribed these drugs, which could be missed if only the parent drugs are detected [47,48]. Advantages of LC–MS/MS for measurement of opioids LC–MS/MS is a very flexible technique that allows the user to develop assays specific to their clinical need. The laboratory developing the assay has control over exactly which opioids to add into the assay and therefore can tailor it to their patient population, with the only limitation being the availability of drug standards and labeled IS. Using an IS (isotopic-labeled) that elutes from the chromatography column at the same time as the analyte of interest can help account for any ion suppression or matrix effects that can occur from analyzing a urine matrix and is therefore strongly recommended, as is evaluation of the degree of ion suppression in the method [49,50]. Using LC–MS/MS affords flexibility in measurement of polar and nonpolar compounds based upon the choice of stationary phase and mobile phases. This is particularly useful in the quantification of opioid metabolites in urine as by definition they are commonly excreted as more polar compounds, for example, the glucuronides. Additionally, LC– MS/MS allows for the ana­lysis of nonvolatile compounds without the need for derivatization, reducing the time taken for sample preparation. Another advantage of LC–MS/MS technology is that it is constantly improving with regard to sensitivity allowing the user to detect analytes at very low concentrations, which may be required in order to quantify parent opioid drugs in urine. future science group

| R eview

The challenges of LC–MS/MS ana­lysis of opiates and opioids in urine Urine sample preparation A major concern in the LC–MS/MS ana­lysis of opioids in urine is sample preparation. This should be sufficient enough to clean up the sample to minimize ion suppression or matrix effects from hydrophilic residual matrix components such as inorganic salts, to concentrate the analytes in the sample if required, and to keep the chromatography column and the mass spectrometer as clean as possible, but simple enough not to limit sample throughput [51]. As already mentioned, a number of opioids are excreted in the urine as glucuronide metabolites and so in order to maximize detection of these drugs in urine, it is important to be able to quantify these metabolites as well as the parent drug. The gold standard in the past was to remove the glucuronide groups by either acid hydrolysis or glucuronidase treatment and then quantify the total parent drug by GC–MS [52]. Since LC–MS/MS has become a widely accepted methodology for toxicology testing, this step is no longer required, which can greatly simplify the sample preparation process [53]. However, there are some reports of glucuronide removal before ana­lysis by LC–MS/ MS using b-glucuronidase extracted from Patella vulgata [54–56] or Helix pomatia [57] (Table 1). In published literature, there are three main sample preparation methodologies employed in the ana­lysis of urine opioids: protein precipitation (PPT) (Table 2), dilution (Table 3) and SPE (Table 4). Three reports document PPT methods where an IS, in methanol or acetonitrile depending on the analyte, is added to the urine sample then vortexed, centrifuged and injected onto the chromatography column [57–59]. Dilution methods vary in the diluent and whether the urine samples are centrifuged before they are diluted or after addition of the diluent. One group reported a strategy where the diluent was methanol/ water (1:1, v/v) and the diluted sample was vortexed then centrifuged [60]. Another group used water:acetonitrile (95:5) as the diluent that was added after the urine was centrifuged [61]. In three different methods, the authors report centrifugation of the urine specimen and then addition of an equal volume of IS in water [48], an equal volume of IS in 20 mM ammonium acetate buffer [62] or the IS in water added at a fourfold higher volume than the urine [63]. The advantage of using PPT or dilution for the sample preparation step is obviously savings in both time and reagent costs. However, a disadvantage is that if the sample is diluted too much some low concentration analytes may be diluted beyond the range of detection. Another future science group

is that the sample is not really being cleaned up before it is injected onto the analytical column. This can cause remaining matrix components to HO

OH O

HO

O

N

OH

N H3C

O

O

HOOC

H3C

O HO

O HO

CH3

Buprenorphine

Buprenorphine-3-glucuronide

HO

O

O

HOOC

OH O

HO

O

NH

OH

NH H3C

H 3C

O HO

CH3 C(CH3)3

C(CH3)3

O CH3

HO

CH3

C(CH3)3

C(CH3)3 Norbuprenorphine

Norbuprenorphine-3-glucuronide

Figure 4. Buprenorphine and its metabolites.

N

O

CH3 N H3C

N+

CH2

CH3 Methadone

CH3

CH3

EDDP

EMDP

HO

O

N

OH

HO O

N

HO

O

HO Naloxone

O H3C

OH

O

O

O

O

OH Naloxone-3-glucuronide

N N

O

N NH

H3C

Fentanyl

Norfentanyl

Figure 5. Methadone, naloxone, fentanyl and selected metabolites.

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French

Table 1. LC–MS/MS methods for determination of urine opioids† using hydrolysis as the sample preparation technique. Opioids

Urine Sample sample preparation volume (µl) technique

Chromatography Mobile column phases‡

Analysis Ionization time method (min)

LOQ (ng/ ml)

Advantages/ disadvantages

50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 2 2 1 1 2

Includes nor-metabolites and measures glucuronides due to hydrolysis. Uses hydrolysis, LOQs are higher than other methods, missing method information

[54]

Includes nor-metabolites and measures glucuronides due to hydrolysis. Uses hydrolysis, LOQs are higher than other methods, missing method information

[55]

Measures glucuronides due to hydrolysis, thorough sample preparation, low LOQs. Uses hydrolysis, includes only a few opioids, long chromatography method, long sample preparation Measures glucuronides due to hydrolysis. Uses hydrolysis, only measures a few opioids, LOQs are higher than other methods

[56]

COD ND NORCOD MOR HDCN DIHYDRCOD NORHDCN HDMN OXYC NOROXYC OXYM COD ND NORCOD MOR HDCN NORHDCN HDMN DIHYDRCOD OXYC NOROXYC OXYM MOR 500 COD 6MAM EDDP METH

Hydrolysis with ND b-glucuronidase

ND

ND

ESI

Hydrolysis with ND b-glucuronidase

ND

ND

ESI

Hydrolysis with Atlantis® dC18 b-glucuronidase followed by SPE

2 mM, pH 3.0 ammonium formate in water; acetonitrile

18

ESI

MOR HDMN COD HDCN

Hydrolysis with Allure® PFP Propyl b-glucuronidase

10 mM 11 ammonium formate, 0.0005% formic acid in water; 10 mM ammonium formate, 0.0005% formic acid in methanol

ESI

1000

60 60 60 60

Ref.

[57]

In some cases, other drugs and metabolites were quantified in the LC–MS/MS methods along with the opioids. All percentages or ratios given here are expressed as volume concentration (v/v). 6MAM: 6-monoacetylmorphine; COD: Codeine; DIHYDRCOD: Dihydrocodeine; EDDP: 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; HDCN: Hydrocodone; HDMN: Hydromorphone; METH: Methadone; MOR: Morphine; ND: Not documented; ND: Not determined; NORCOD: Norcodeine; NORHDCN: Norhydrocodone; NOROXYC: Noroxycodone; OXYC: Oxycodone; OXYM: Oxymorphone. † ‡

elute with the analyte of interest into the mass spectrometer causing a change in the ionization efficiency, or these components could be retained on the column and elute at a later time. Therefore, matrix effects or ion suppression could reduce the sensitivity and precision of the method and so should be investigated, as should potential interferences [51,64]. 2808

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SPE involves adding the urine sample to a column or plate containing a stationary phase or sorbent that has specific properties that should either retain the analytes of interest and allow the rest of the urine matrix components to flow through, or allow the analytes to flow through and retain the matrix components. If the analytes are retained, they can be washed on the sorbent future science group

The challenges of LC–MS/MS ana­lysis of opiates and opioids in urine to remove any more matrix components before being eluted and then used for LC–MS/MS ana­lysis. A number of different SPE stationary phase sorbents exist such as those employing neutral, anion exchange and cation exchange mechanisms. Specifically for opioid ana­lysis, a number of published reports utilize cation exchange SPE sorbents [47,65–68] whereas other authors used balanced hydrophilic and lipophilic

| R eview

SPE sorbents [56] or hydrophobic SPE sorbents [69,70]. The advantage of using SPE as the sample preparation step is that it cleans up the matrix more effectively than both PPT or dilution and this can reduce potential matrix effects, which will increase the sensitivity and precision of the LC–MS/MS method [64]. However, once the analytes are eluted from the SPE sorbent, they are commonly dried down and reconstituted in a

Table 2. LC–MS/MS methods for determination of urine opioids† using protein precipitation as the sample preparation technique. Opioids

Urine Sample Chromatography Mobile sample preparation column phases‡ volume (µl) technique

Analysis time (min)

Ionization LOQ method (ng/ ml)

Advantages/ disadvantages

NORMOR MOR NORCOD COD 6MAM HEROIN 6ACOD PAPAV METHADOL METH EDDP

100

Protein precipitation

Synergi™ Polar RP

35

APCI

M-3-G M-6-G NORMOR C-6-G MOR 6MAM COD NORCOD HEROIN 6ACOD PAPAV NOSCAP

100

Protein precipitation

Synergi™ Fusion RP 10 mM ammonium acetate in water; acetonitrile; methanol

Parent drugs: ESI 35; glucuronides: 15

25 25 50 25 50 10 25 25 10 10 10 10

MOR COD M-3-G M-6-G C-6-G

200

Protein precipitation

Synergi Fusion RP

15

25 25 25 25 25

Measures normetabolites, simple sample preparation, small urine sample volume. Does not measure glucuronides, less thorough sample preparation, long chromatography method, some LOQs are higher than other methods Measures glucuronides and nor-metabolites, small urine sample volume, simple sample preparation. Parent drugs and glucuronides are measured in two different methods, less thorough sample preparation, long chromatography method, LOQs are higher than other methods Includes glucuronides, low urine sample volume, simple sample preparation. Does not include many opioids or nor-metabolites, less thorough sample preparation, long chromatography method

10 mM ammonium formate, 0.001% formic acid in water; acetonitrile

10 mM ammonium acetate in water; acetonitrile; methanol

ESI

50 50 25 25 10 10 10 10 100 10 10

Ref.

[58]

[59]

[65]

In some cases, other drugs and metabolites were quantified in the LC–MS/MS methods along with the opioids. All percentages or ratios given here are expressed as volume concentration (v/v). 6ACOD: 6-acetylcodeine; 6MAM: 6-monoacetylmorphine; C-6-G: Codeine-6b -glucuronide; COD: Codeine; EDDP: 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; M-3-G: Morphine-3b -glucuronide; M-6-G: Morphine-6b -glucuronide; METH: Methadone; METHADOL: Methadol; MOR: Morphine; NORCOD: Norcodeine; NORMOR: Normorphine; NOSCAP: Noscapine; PAPAV: Papaverine. † ‡

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French smaller volume of solvent or buffer, which in fact concentrates both the analyte and the remaining matrix. This can actually increase the matrix effects, although not to as great a degree as seen in PPT or diluted samples [51]. Another less commonly reported sample preparation methodology is online extraction (Table 5) where in one report, the urine sample was first

diluted with methanol and water, IS added, and samples vortexed and injected onto an online trap column where they were washed in 10 mM ammonium acetate in water before injection onto the chromatography column [71]. A different group reported use of a balanced hydrophilic and lipophilic online extraction column after the urine was diluted with 0.01% trifluoroacetic acid

Table 3. LC–MS/MS methods for determination of urine opioids† using dilution as the sample preparation technique. Opioids

Urine Sample Chromatography Mobile phases‡ sample preparation column volume technique (µl)

Analysis Ionization LOQ time method (ng/ (min) ml)

M-3-G 100 OXYM-3-G HDMN-3-G M-6-G MOR OXYM HDMN C-6-G COD OXYC 6MAM HDCN NBUP-3-G NORFENT MEPIR NORMEPIR FENT BUP METH PROPOX MOR 100 COD DIHYDRCOD 6MAM M-3-G EDDP BUP

Dilution in Acquity UPLC® HSS equal volume T3 column of IS in water

2 mM ammonium 9 acetate, 0.1% formic acid in water; 0.1% formic acid in acetonitrile

ESI

Dilution with HyPURITY™ C8 equal volume of 50:50 methanol: water

25 mM ammonium 12.5 acetate in 95:5 water:methanol; 0.05 mM formic acid in 98:2 methanol: propan-2-ol

ESI

M-3-G M-6-G MOR OXYM HDMN NORCOD COD OXYC 6MAM HDCN

Dilution with 95:5 water: acetonitrile

10 mM ammonium 6 formate, 0.05% formic acid in 95:5 water:acetonitrile; 10 mM ammonium formate, 0.05% formic acid in 95:5 acetonitrile:water

ESI

1000

Zorbax® SB Phenyl

0.8 4.9 0.9 0.9 2.5 1.3 1.8 2.6 1.5 1.7 0.6 1.8 2.5 1.4 1.9 1.7 0.05 3.6 2.8 2.5 0.1 0.1 0.1 0.1 0.1 0.1 0.1 50 50 50 50 50 50 50 50 0.25 50

Advantages/ disadvantages

Ref.

Includes a large number of opioids, uses very small urine volume, simple sample preparation, low LOQs. Less thorough sample preparation

[48]

Small sample volume, simple sample preparation, low LOQs. Does not measure glucuronides or nor-metabolites, less thorough sample preparation Measures glucuronides and normetabolites, simple sample preparation, short chromatography method. Less thorough sample preparation, LOQs are higher than other methods

[60]

In some cases, other drugs and metabolites were quantified in the LC–MS/MS methods along with the opioids. All percentages or ratios given here are expressed as volume concentration (v/v). 6MAM: 6-monoacetylmorphine; BUP: Buprenorphine; C-6-G: Codeine-6b -glucuronide; COD: Codeine; DIHYDRCOD: Dihydrocodeine; EDDP: 2-ethylidene-1,5dimethyl-3,3-diphenylpyrrolidine; ETM: Ethylmorphine; ETM-6-G: Ethylmorphine-6b-glucuronide; FENT: Fentanyl; HDCN: Hydrocodone; HDMN: Hydromorphone; HDMN-3-G: Hydromorphone-3b -glucuronide; M-3-G: Morphine-3b -glucuronide; M-6-G: Morphine-6b -glucuronide; MEPIR: Mepiridine; METH: Methadone; MOR: Morphine; NBUP: Norbuprenorphine; NBUP-3-G: Norbuprenorphine-3 b -glucuronide; ND: Not determined; NORCOD: Norcodeine; NORFENT: Norfentanyl; NORMEPIR: Normepiridine; NOROXYC: Noroxycodone; NOROXYM: Noroxymorphone; OXYC: Oxycodone; OXYM: Oxymorphone; OXYM-3-G: Oxymorphone-3 b glucuronide; PROPOX: Propoxyphene. † ‡

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[61]

The challenges of LC–MS/MS ana­lysis of opiates and opioids in urine

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Table 3. LC–MS/MS methods for determination of urine opioids† using dilution as the sample preparation technique (cont.). Opioids

Urine Sample Chromatography Mobile phases‡ Analysis Ionization LOQ sample preparation column time method (ng/ volume technique (min) ml) (µl)

Advantages/ disadvantages

HDMN-3-G MOR-3/6-G NOROXYM OXYM MOR NOROXYC C-6-G HDMN ETM-6-G OXYC COD NORFENT 6MAM ETM EDDP NBUP METH FENT BUP M-3-G MOR M-6-G COD C-6-G 6MAM ETM ETM-6-G

100

Dilution with BEH shield RP18 20 mM ammonium acetate buffer

10 mM ammonium acetate in water; methanol

7.5

ESI

ND

Measures large number of opiods including glucuronides and nor-metabolites, low urine sample volume, simple sample preparation. Less thorough sample preparation, LOQs are not documented

[62]

20

Dilution with water

25 mM formic 13 acid in 99:1 water: acetonitrile; 25 mM formic acid in 90:10 acetonitrile:water

ESI

1 32 71 6 60 10 126 31

Includes glucuronides, very low urine sample volume, simple sample preparation Does not include many opioids or normetabolites, less thorough sample preparation, some LOQs are higher than other methods

[63]

Luna® C18

Ref.

In some cases, other drugs and metabolites were quantified in the LC–MS/MS methods along with the opioids. All percentages or ratios given here are expressed as volume concentration (v/v). 6MAM: 6-monoacetylmorphine; BUP: Buprenorphine; C-6-G: Codeine-6b -glucuronide; COD: Codeine; DIHYDRCOD: Dihydrocodeine; EDDP: 2-ethylidene-1,5dimethyl-3,3-diphenylpyrrolidine; ETM: Ethylmorphine; ETM-6-G: Ethylmorphine-6b-glucuronide; FENT: Fentanyl; HDCN: Hydrocodone; HDMN: Hydromorphone; HDMN-3-G: Hydromorphone-3b -glucuronide; M-3-G: Morphine-3b -glucuronide; M-6-G: Morphine-6b -glucuronide; MEPIR: Mepiridine; METH: Methadone; MOR: Morphine; NBUP: Norbuprenorphine; NBUP-3-G: Norbuprenorphine-3 b -glucuronide; ND: Not determined; NORCOD: Norcodeine; NORFENT: Norfentanyl; NORMEPIR: Normepiridine; NOROXYC: Noroxycodone; NOROXYM: Noroxymorphone; OXYC: Oxycodone; OXYM: Oxymorphone; OXYM-3-G: Oxymorphone-3 b glucuronide; PROPOX: Propoxyphene. † ‡

containing the IS and centrifuged [72]. A third method documented use of an anion exchange online extraction column after dilution of the urine with acetate buffer, addition of IS, b-glucuronidase treatment and centrifugation [73]. Online extraction has a number of advantages that mimic those of SPE in the context of cleaning up the sample and thereby reducing matrix effects. A major advantage of online extraction over SPE is the reduction in hands-on time since the instrument is programmed to carry out all the conditioning and washing steps. A potential disadvantage could be the increased cost of the initial equipment purchase to be able to implement online extraction techniques. future science group

„„LC

columns The ana­lysis of both opioids and their metabolites by LC–MS/MS can be challenging since the parent compounds are nonpolar and the metabolites are polar. Therefore, choice of analytical chromatography column is crucial especially if these compounds are going to be measured in the same run without removing the glucuronide groups before ana­lysis. There are two main types of chromatography that have been used for opioid ana­ lysis: reversed-phase chromatography (RPC) and HILIC, a form of normal-phase chromatography [74]. Far and away the most commonly used type of chromatography for opioid ana­lysis is RPC. In RPC, the stationary phase is hydrophobic and www.future-science.com

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Table 4. LC–MS/MS methods for determination of urine opioids† using SPE as the sample preparation technique. Opioids

Urine Sample Chromatography Mobile phases‡ sample preparation column volume technique (µl)

Analysis Ionization LOQ time method (ng/ (min) ml)

Advantages/ disadvantages

Ref.

MOR M-3/6-G COD C-6-G HDMN HDMN3-G HDCN OXYM OXYM3-G OXYC

1000

SPE

XBridge Amide HILIC

10 mM ammonium 17 formate, 0.125% formic acid in 50:50 acetonitrile:water; 10 mM ammonium formate, 0.125% formic acid in 90:10 acetonitrile:water

ESI

1.25 1.25 0.31 0.31 0.31 0.31 0.16 2.5 0.63 0.31

Includes glucuronides, thorough sample preparation, low LOQs does not include many opioids or normetabolites, measures M-3-G and M-6-G together, long sample preparation, long chromatography method

[47]

MOR HDMN COD OXYC 6MAM HDCN

1000

SPE

Nova-Pak CN HP

Isocratic elution using 2 mM ammonium formate, pH 3.0 in 85:15 water:acetonitrile

10

ESI

2 2 2 2 2 2

Thorough sample preparation, low LOQs; does not include glucuronides or normetabolites, long sample preparation

[66]

MOR COD 6MAM PHOL OXYC ETM

500

SPE

Acquity UPLC® BEH pH 10.2, 5 mM C18 ammonium bicarbonate in water; methanol

5.7

ESI

7.9 7.0 3.2 64.0 5.0 6.5

Short chromatography method; does not include glucuronides or nor-metabolites, long extraction method

[67]

BUP NBUP BUP-3-G NBUP3-G NAL

1000

SPE

Acquity UPLC BEH C18

pH 3.0, 5 mM ammonium formate in water; 0.05% formic acid in methanol

ND

ESI

2 2 5 5 50

Includes glucuronides and nor-metabolites, thorough sample preparation, low LOQs; does not include may opioids, long sample preparation

[68]

BIS 1000 BUP COD FENT HDMN M-3-G M-6-G METH MOR NORFENT NORTIL OXYC OXYM PIRITR TIL TRAM

SPE

C12 MAX-RP

pH 3.0, 5 mM ammonium formate in 90:10 water:acetonitrile; pH 3.5, 5 mM ammoinium formate in 90:10 acetonitrile:water

35

ESI

0.9 1.7 4.9 0.2 1.6 6.6 8.4 2.3 4.7 0.2 1.1 5.3 2.9 2.6 0.8 3.0

Includes large number of opioids and some glucuronides and normetabolites, thorough sample preparation, low LOQs; long sample preparation, long chromatography method

[69]

In some cases, other drugs and metabolites were quantified in the LC–MS/MS methods along with the opioids. All percentages or ratios given here are expressed as volume concentration (v/v). 6MAM: 6-monoacetylmorphine; BIS: Bisnortilidine; BUP: Buprenorphine; BUP-3-G: Buprenorphine-3b -glucuronide; C-6-G: Codeine-6b-glucuronide; COD: Codeine; ETM: Ethylmorphine; FENT: Fentanyl; HDCN: Hydrocodone; HDMN: Hydromorphone; HDMN-3-G: Hydromorphone-3b-glucuronide; M-3-G: Morphine-3b glucuronide; M-6-G: Morphine-6b -glucuronide; METH: Methadone; MOR: Morphine; NAL: Naloxone; NBUP: Norbuprenorphine; NBUP-3-G: Norbuprenorphine-3 b glucuronide; ND: Not determined; NORFENT: Norfentanyl; NORMOR: Normorphine; NORTIL: Nortilidine; OXYC: Oxycodone; OXYM: Oxymorphone; OXYM-3-G: Oxymorphone-3b -glucuronide; PHOL: Pholcodine; PIRITR: Piritramide; TIL: Tilidine; TRAM: Tramadol. † ‡

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future science group

The challenges of LC–MS/MS ana­lysis of opiates and opioids in urine

| R eview

Table 4. A review of published LC–MS/MS methods for determination of urine opioids† using SPE as the sample preparation technique (cont.). Opioids Urine Sample Chromatography Mobile phases‡ Analysis Ionization LOQ sample preparation column time method (ng/ volume (µl) technique (min) ml)

Advantages/ disadvantages

M-3-G 1000 NORMOR MOR COD 6MAM METH

Includes glucuronide and nor-metabolite, thorough sample preparation, low LOQs; does not measure M-6-G, does not include many opioids, long sample preparation, long chromatography method

SPE

Synergi™ Hydro-RP pH4.6, 4 mM ammonium acetate in water: acetonitrile

23

ESI

6.6 8.0 6.3 3.1 5.3 1.8

Ref.

[70]

In some cases, other drugs and metabolites were quantified in the LC–MS/MS methods along with the opioids. All percentages or ratios given here are expressed as volume concentration (v/v). 6MAM: 6-monoacetylmorphine; BIS: Bisnortilidine; BUP: Buprenorphine; BUP-3-G: Buprenorphine-3b -glucuronide; C-6-G: Codeine-6b-glucuronide; COD: Codeine; ETM: Ethylmorphine; FENT: Fentanyl; HDCN: Hydrocodone; HDMN: Hydromorphone; HDMN-3-G: Hydromorphone-3b-glucuronide; M-3-G: Morphine-3b glucuronide; M-6-G: Morphine-6b -glucuronide; METH: Methadone; MOR: Morphine; NAL: Naloxone; NBUP: Norbuprenorphine; NBUP-3-G: Norbuprenorphine-3 b glucuronide; ND: Not determined; NORFENT: Norfentanyl; NORMOR: Normorphine; NORTIL: Nortilidine; OXYC: Oxycodone; OXYM: Oxymorphone; OXYM-3-G: Oxymorphone-3b -glucuronide; PHOL: Pholcodine; PIRITR: Piritramide; TIL: Tilidine; TRAM: Tramadol. † ‡

the mobile phase is polar allowing hydrophobic analytes to be retained by the column [75]. In other words, using RPC, the more nonpolar compounds such as morphine will interact with the column and move slowly along it, and the more polar compounds such as the glucuronide metabolites will not interact to as great an extent with the column, and therefore will be eluted quicker [75]. In HILIC, the stationary phase is hydrophilic and the mobile phase is less polar allowing polar analytes such as the glucuronide metabolites to be retained by the column and the more nonpolar analytes such as morphine to elute more quickly as they do not interact as considerably with the column [47,74]. A vast array of stationary-phase chemistries are available for RPC and a large number of different kinds have been used in published opioid LC–MS/MS methods. Reversed-phase column stationary phases that have been used in opioid ana­lysis include high-strength silica [48], difunctionally bonded ligands [56], pentafluoro­phenyl phase with propyl spacer [57], phenyl columns [58,61], a C8 column [60], C18 columns with carbamate [62], a phenyl phase attached to a C6 ligand [70], hydrophilic endcapping [63,71–73], polar embedded groups [65], ethylene bridged hybrid technology [67,68], a cyano column [66], and a C12 hydrophobic end-capped column [69]. A number of these columns are designed to allow ana­lysis of both polar and nonpolar compounds, which is extremely useful in the ana­lysis of opioids and their metabolites. As the polar compounds elute early from RPC columns, it is future science group

important to ensure that they do not elute in an area of ion suppression due to the matrix eluting at the same time. One way of helping account for this is to use a labeled IS for each compound in the method, but this is not always possible due to the lack of commercial availability of these IS and the high cost of m­anufacturing them as custom products. HILIC columns have not been used frequently in published LC–MS/MS methods for opioid ana­lysis, although they have unique selectivity for polar analytes, and thus are worth mentioning [74]. One author reported using a HILIC stationary phase that consisted of an ethylene bridged hybrid particle coupled with an amide phase, in which the opioid glucuronide metabolites were significantly retained on the column and the less polar drugs such as morphine, hydromorphone, hydrocodone and oxycodone were eluted from the column earlier when starting in an acetonitrilebased mobile phase [47]. Since the gradient for HILIC starts in high organic solvent concentration, the analytes are usually injected in a highly organic solution, which can reduce the requirement for drying down and reconstituting the sample after PPT or SPE [74]. This obviously saves time, but can also increase the sensitivity of the LC–MS/MS method and reduces the possibility of mismatching the reconstitution solution, which can have a large effect on the chromatographic peak shape. One limitation of using a HILIC column is that they tend to require a longer equilibration than RPC columns and this can increase ana­lysis time [76,77]. www.future-science.com

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Table 5. A review of published LC–MS/MS methods for determination of urine opioids† using online extraction as the sample preparation technique. Opioids

Urine Sample sample preparation volume technique (µl)

Chromatography Mobile phases‡ column

Analysis time (min)

COD C-6-G MOR M-3-G M-6-G 6MAM METH EDDP BUP NBUP BUP-3-G NBUP-G

100

Online extraction

AQUASIL C18

10 mM ammonium acetate in water; 10 mM ammonium acetate in 50:50 methanol: acetonitrile

15 ESI (including online extraction)

2000

Dilution with 0.01% TFA followed by online extraction

SymmetryShield™ RP18

0.05% TFA in 95:5 water:acetonitrile; 0.05% TFA in 95:5 acetonitrile:water

19 ESI (including online extraction)

MOR 500 COD DIHYDRCOD 6MAM EDDP BUP NBUP TIL NORTIL TRAM DEMETHTRAM

Dilution with Hypersil GOLD acetate buffer, aQ™ hydrolysis with b-glucuronidase followed by online extraction

Ionization LOQ method (ng/ ml)

0.1% formic acid in 10 min ESI water; 0.1% formic (including acid in acetonitrile online extraction)

25 25 25 25 25 25 15 15 0.5 1.0 0.5 0.5

5.0 9.2 2.7 ND 3.6 2.3 4.2 8.4 3.2 5.6 7.5

Advantages/ Ref. disadvantages

Includes glucuronides, small urine sample volume, online extraction. LOQs higher than other methods Includes glucuronides and nor-metabolites, online extraction, low LOQs. Only measures a few opioids, largest urine sample volume Includes glucuronides due to hydrolysis, online extraction, low LOQs. Uses hydrolysis increasing extraction method time

[71]

[72]

[73]

In some cases, other drugs and metabolites were quantified in the LC–MS/MS methods along with the opioids. All percentages or ratios given here are expressed as volume concentration (v/v). 6MAM: 6-monoacetylmorphine; BUP: Buprenorphine; BUP-3-G: Buprenorphine-3b -glucuronide; C-6-G: Codeine-6b-glucuronide; COD: Codeine; DEMETHTRAM: O-demthyltramadol; DIHYDRCOD: Dihydrocodeine; EDDP: 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; M-3-G: Morphine-3b-glucuronide; M-6-G: Morphine6b -glucuronide; METH: Methadone; MOR: Morphine; NBUP: Norbuprenorphine; NBUP-3-G: Norbuprenorphine-3b-glucuronide; NORTIL: Nortilidine; TFA: Trifluoracetic acid; TIL: Tilidine; TRAM: Tramadol. † ‡

LC mobile phases As mentioned above, the majority of published LC–MS/MS methods for opioid ana­lysis use RPC. All but one of the reports using RPC reviewed here use gradient elution and therefore the mobile phases used were more water-based for the starting conditions ramping to predominantly organic-based in order to elute the opioids from the RPC column (Table 1). The one report of isocratic elution used a constant flow of mobile phase consisting of ammonium formate in a mixture of water and acetonitrile [61]. The most commonly used buffer was ammonium formate followed by ammonium acetate. Formic acid was added in nearly half of the reviewed methods and 0.05% trifluoroacetic acid was used in one method [72]. By far the most common organic solvent used was acetonitrile, but 2814

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methanol and propan-2-ol were used in one method [60], and methanol was used both alone and in combination with acetonitrile. In the only HILIC method reviewed here, gradient elution was used and the mobile phases were composed of ammonium formate and formic acid in a m­ixture of water and acetonitrile [47]. Published LC–MS/MS methods As discussed, there are a large number of opioid compounds that are prescribed, taken illegally or formed from metabolism of these ingested compounds. Unsurprisingly, there are no published LC–MS/MS methods that include all of these compounds especially given the complex data ana­lysis that would be required, as well as a potentially limiting long chromatography method. A review of the literature uncovered future science group

The challenges of LC–MS/MS ana­lysis of opiates and opioids in urine a number of LC–MS/MS methods that vary with regard to the opioids included, the required urine sample volume, the length of the chromatography run and the LOQ of these compounds (Tables 1–5). Only two out of the 21 published LC–MS/MS methods reviewed did not include quantification of morphine and codeine, which is unsurprising. The two methods that did not quantify morphine and codeine appear to have been developed for the specific purpose of quantifying buprenorphine and its metabolites [68,72]. Surprisingly, even though hydrocodone is the most prescribed opioid in the USA and is heavily prescribed in other countries, this opioid was only quantified in a third of the reviewed methods [47,48,54,55,57,61,66]. Hydromorphone and oxycodone were quantified in approximately 38 and 43% of the reviewed published methods, respectively, and oxymorphone was quantified in approximately 33% of the reviewed papers. A number of laboratories are obviously serving clinicians that are concerned about their patients taking heroin as in 62% of the reviewed published methods, 6MAM was included. In two published LC–MS/MS methods, heroin itself was included, as well as 6-acetylcodeine, an impurity found in illicit preparations of heroin [58,59]. Additionally, a number of laboratories must accommodate samples from addiction specialists as 52% of the reviewed methods quantify either methadone, EDDP (methadone m­etabolite) or buprenorphine and its metabolites. It has been reported that only measuring parent drug and not the glucuronide metabolites in urine can lead to false-negative results for opioids [47,59]. In the published reports reviewed here, the vast majority either quantified one or more glucuronide metabolites, or used b-glucuronidase to remove the glucuronide groups before ana­lysis in order to measure the total drug. Although using b-glucuronidase aids in the sensitivity of the method with regard to detecting total drug, it can also increase the variability in the method and therefore affect precision due to incomplete or inconsistent enzymatic hydrolysis. Interestingly, in the methods that quantified glucuronide metabolites without use of glucuronidase, two only quantified morphine-3b-glucuronide and not morphine-6b-glucuronide, the active glucuronide metabolite [60,70]. Additionally, two reports combined quantification of the morphine3b-glucuronide and morphine-6b-glucuronide metabolites instead of quantifying them independently [47,62]. Since the majority of morphine is excreted in the morphine-3b-glucuronide form, future science group

it seems as though it would be most important to quantify this metabolite, especially since the concentrations will remain higher for longer in the urine, extending the detection window [15]. Three of the reviewed reports did not quantify any glucuronide metabolites, nor did they utilize glucuronidase in order to quantify total drug [58,66,67]. Although Dams et al. did not quantify the glucuronide metabolites, they did quantify the N-demethylated metabolites; normorphine and norcodeine [58]. It has been shown that only approximately 5% of a morphine dose is converted to normorphine and in one study, only a trace of norcodeine was found in the urine of codeine users [15]. However, in one report, approximately 8.6% of urine specimens that were negative for codeine were positive for norcodeine, illustrating that inclusion of this metabolite can aid in monitoring codeine use [55]. With respect to the N-demethylated metabolites, approximately 57% of the reviewed reports include at least one of these metabolites in the LC–MS/MS method. The most commonly quantified one is nor­ codeine followed by normorphine, n­oroxycodone, no­rfentanyl and norbuprenorphine. Two published LC–MS/MS methods were specifically designed for the detection of the semisynthetic opioid buprenorphine and its metabolites buprenorphine-3b-glucuronide, norbuprenorphine and norbuprenorphine-3b-glucuronide [68,72]. As mentioned previously, this opioid is often given as part of treatment for opioid dependence and so compliance with this treatment is often monitored. The inclusion of metabolites in the monitoring of buprenorphine compliance is important as only a very small percentage of parent drug is found in urine [78]. In one report, it was found that approximately 20% of buprenorphine samples were positive when measured by a LC–MS/MS method that included the glucuronide metabolite, but were negative when screened with an immuno­assay method – indicating that this immunoassay method may only be specific for the parent drug [48]. This point is extremely important as noncompliance with medication is a reason for discontinuing the prescribing of drugs such as buprenorphine in opioid dependence treatment, and so a patient could be documented as noncompliant and potentially removed from the treatment program even though they are a­ctually taking their medication as prescribed. Propoxyphene was only included in one LC–MS/MS method, perhaps due to the fact that it has been removed from the market in both Europe and the USA [48]. Some of the www.future-science.com

| R eview

Key Term Compliance: The act of taking opioid drugs as and when prescribed by a clinician.

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French less frequently quantified opioids in the methods reviewed here include two opium alkaloids papaverine and noscapine (nonaddictive; can be found in heroin; used as an antitussive; no pain-killing properties), the semi-synthetic opioid dihydrocodeine, and the synthetic opioids and their metabolites naloxone, fentanyl/norfentanyl, mepiridine/normepiridine (pethidine), methadol (related to methadone; active ingredient of levo-a-acetylmethadol), ethylmorphine/ ethylmorphine-6b-glucuronide, pholcodine (antitussive), tilidine/bisnortilidine/nortilidine (commonly used in Germany), piritramide (commonly used in The Netherlands, Denmark and Germany) and tramadol/O-demethyl­ tramadol. The inclusion of these opioids in a LC–MS/MS method is obviously dependent upon the prevalence of use in the country where the method will be carried out. In all but one of the reviewed methods [58], ESI was utilized for the ana­lysis of opioids by LC–MS/MS and every method used positive polarity. APCI was utilized in the other method [58], and although the authors did not specifically state why they chose APCI over ESI, this type of ionization has been shown to have reduced matrix effects or ion suppression by the same group [51] and it can be useful in the quantification of nonpolar compounds [79]. Additionally, all of the methods used SRM for quantification of the opioids where a precursor ion of a particular mass is selected in the first quadrupole of the mass spectrometer and a product ion is selected in the third quadrupole of the mass spectrometer after fragmentation in the second quadrupole. Five of the reviewed MS methods utilized two SRM transitions for each analyte and for each IS: a quantifier transition and a qualifier transition [47,48,66–68]. This is the best practice scenario for any LC–MS/MS method as it allows for the use of ion ratios whereby the ratio of the peak areas of the two different transitions can be monitored to allow for detection of potential interferences in the method, which could skew these ratios and affect quantitation. Eight of the 21 reviewed methods utilized two SRM transitions for the analytes and one SRM transition for the IS [54–56,60,62,63,69,73]. An advantage of this approach is that it reduces data ana­lysis time, but a disadvantage is that potential interferences in the IS transition may not be seen, which could potentially affect quantification of the opioids. In two of the methods, one SRM transition was used for both the analytes and

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the IS [61,72] and in the other methods, at least one SRM transition was used for the analytes and the IS [58,59,65,70], another utilized SRM followed by ana­lysis of a full scan product ion spectra to identify opioids that were present as determined by isotope dilution relative to the labeled IS [57] and in one report the authors did not document how the IS were monitored [71]. One parameter that does vary significantly between the published LC–MS/MS methods is the LOQ for the opioids. It must be noted, that a number of these published assays also detect other drug classes such as amphetamines and cocaine, so the LC–MS/MS parameters may not be completely optimized for detection of o­pioids. Additionally, as mentioned before, some of these methods used PPT or dilution strategies to minimize sample preparation, which could reduce the sensitivity of the method both by diluting down some analytes, or by the presence of ion suppression or matrix effects. In clinical laboratories, most LC–MS/MS assays would be utilized for confirmation of a positive opiate immunoassay screen, which would more than likely be most specific for morphine and have less crossreactivity to the rest of the opiates and o­ pioids, although other individual immuno­assays exist that are specific for 6MAM, oxy­codone and buprenorphine, they are not too wid­ely adopted. As such, the cut-off in which a urine sample is documented as positive by LC–MS/MS will be a lower concentration than that of the immuno­a ssay screening method. The commonly used opioid immuno­assay cut-off concentration is 300 ng/ml for routine clinical toxicology testing [43] and for oxycodone immunoassays, the cut-off concentration is 100 ng/ml. All of the LC–MS/MS assays reviewed here have LOQs that are significantly below this cut-off, indicating they would all be appropriate for clinical confirmatory testing. One of the published methods documented using a 10 ng/ml cut-off concentration for the opioids in the LC–MS/MS method and this gave the same number of positive patient urine samples as the GC–MS comparison method [47]. In another method, the cut-off was 10 ng/ml for the parent compounds and 50 ng/ml for the glucuronides [48]. These cut-off concentrations were above the LOQ documented for these LC–MS/MS assays, but for the remaining reviewed methods, the LOQ was used as the cut-off concentration, which varied from 0.1 to 126 ng/ml depending on the method and the analyte in question. future science group

The challenges of LC–MS/MS ana­lysis of opiates and opioids in urine The chromatography run time in these reviewed methods varies from 6 to 35 min, although for three of the reports the length of the chromatography method was not documented [54,55,68]. Compared with published LC–MS/MS methods for detection of other compounds, these run times are fairly extensive. However, as mentioned previously, given how structurally similar a number of the opioids are, they require chromatographic separation from each other before they enter the MS, and this can add some time to the method. One of the LC–MS/MS methods actually quantified 19 opioids along with 111 other drugs in a 7.5 min method with simple PPT, but only one IS was utilized and the LOQ for each compound was not determined, although the LOD was determined and a drug was not reported as present if the peak was below the LOD (defined as a signal to noise ratio [S/N] of 3:1) [62]. The volume of urine required for ana­lysis in all of these methods varies significantly from 20 up to 2000 µl, although the volume is not documented in two of the reviewed methods. Under normal circumstances, voided urine volume is not usually a limiting factor in drug ana­lysis, but in some particularly sick patients it could be a problem; thus, limiting the required sample volume while retaining the sensitivity of the method could be important. Future perspective As online extraction methodologies become more common place, the cost will decrease allowing clinical laboratories to implement these, thereby reducing the hands-on time required for opioid

| R eview

assays. Continual improvements in chromatography column technology means that separating isobaric and isomeric opioids will become easier, enabling faster run times to be obtained, increasing throughput in the laboratory. Given that the US population makes up approximately 99% of the world hydrocodone consumption, urine opioid LC–MS/MS assays serving this population should be able to quantify hydrocodone and norhydrocodone and potentially hydromorphone and hydromorphone-3b-glucuronide, especially since current opiate immunoassay methodologies have only partial crossreactivity with these compounds. With the advent of more clinical laboratories implementing MS testing, the availability of labeled IS for the opioids and the metabolites should increase enabling more precise quantification of all of these compounds. Since LC–MS/MS methods are proving less and less time consuming as far as sample preparation goes, and more affordable with regard to reagent costs, ultimately they could replace immunoassays as a screening method as they have superior specificity and sensitivity, especially when it comes to the complex opioids. Financial & competing interests disclosure The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert t­estimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Executive summary Use & abuse of opioids „„

Opiate and opioid prescribing and abuse has significantly increased in the past 20 years, partly due to the increased recognition of chronic pain as a major problem in the general population.

„„

Clinicians have both a clinical and legal obligation to monitor opioid use when prescribing opioids.

Metabolism of opioids „„

The metabolism of opioids is extremely complex and should be understood by the clinician prescribing opioids and the laboratory performing the urine drug testing.

Accurate quantification of opioids is necessary „„

Standard of clinical care is to run a urine immunoassay drug screen and then confirm any positive results by a more specific methodology.

„„

GC–MS was the gold standard for confirmatory testing, but LC–MS/MS is overtaking GC–MS as the method of choice.

Advantages of LC–MS/MS for measurement of opioids „„

The advantages of LC–MS/MS for this application are that it does not require derivatization; removal of the glucuronide groups is not required as they can be quantified intact; and, it is capable of detecting both nonpolar and polar compounds in one run and, therefore, sample preparation time can be greatly reduced increasing assay throughput.

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References Papers of special note have been highlighted as: nn of considerable interest 1

2

Matthes HW, Maldonado R, Simonin F et al. Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the µ-opioid receptor gene. Nature 383, 819–823 (1996). Fricchione GL, Mendoza A, Stefano GB. Morphine and its psychiatric implications. Adv. Neuroimmunol. 4(2), 117–131 (1994).

3

Pattinson KT. Opioids and the control of respiration. Br. J. Anaesth. 100(6), 747–758 (2008).

4

Manara L, Bianchi G, Ferretti P, Tavani A. Inhibition of gastrointestinal transit by morphine in rats results from direct drug action on gut opioid sites. J. Pharmacol. Exp. Ther. 237(3), 945–949 (1986).

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MS analysis of opiates and opioids in urine.

Opioids are some of the most commonly prescribed and abused drugs around the world. Primarily used for anesthesia or pain management, other opioids ca...
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