Methods

The ranges used for the detection of drug analytes have lower limits and upper limits known as the lower limit of quantification (LLOQ) and the upper limit of quantification (ULOQ) shown in Table 3. These levels were determined based on method validation, sensitivity of the instruments, and the ability of the assay to reduce false negative results significantly compared to SAMHSA levels.1

The purpose of a lower limit is to prevent false negative results, while the upper limit is defined by the range of the calibration curve. Concentrations of an analyte that fall below the LLOQ are reported as “negative,” while those within the range or above the ULOQ are reported as “positive” meaning the analyte is present in that sample.

An internal standard (IS) is used in this assay for the determination of accurate unknown concentrations and is highly important for an assay that tests over 100 analytes. The analytes used are labelled by substituting hydrogens (1H) with deuterium (2H), carbon-12 with carbon-13 (13C), or nitrogen-14 with nitrogen-15 (15N) for separate detection from the true analytes ( Table 4). They have been selected from the full list of analytes, and represent frequently used drugs that elute from the column early or where there are regions of many of analytes eluting at similar relative retention times.1

The analytes that were found to have the greatest matrix effects were also chosen for the internal standard in order to compensate for ion suppression or enhancement to still allow for reproduceable quantification. The internal standard helps differentiate between drug analytes that elute at the same retention time, although one downside of IS use is a decrease in the sensitivity in areas of the ion chromatogram that have a high density of analyte peaks.1 Internal standard is added to every sample as well as each calibrator and quality control during extraction, with the exception of a double blank that has no concentration of analytes as well as no IS in the blank urine matrix.

Table 3: LLOQ and ULOQ (ng/mL)

DRUG CLASS

DETECTION RANGES (ng/mL)

Analyte

LLOQ

ULOQ

Analgesic
Ibuprofen
Acetaminophen
Naproxen
Tapentadol
Tramadol
-       O-Desmethyl-cis-Tramadol (metabolite)

 

250
100
10
10
10
10

 

5000
1000
1000
1000
1000
1000

Anesthetics
Ketamine
-       Norketamine (metabolite)
Lidocaine

 

10
10
10

 

1000
1000
1000

Anticonvulsants
Topiramate
Gabapentin
Lamotrigine
Pregabalin

 

10

10
50

 

1000
1000
1000
1000

Antidepressants
1-(3-Chlorophenylpiperazine)
Amitriptyline
-       Nortriptyline (Metabolite)
Aripiprazole
Bupropion
Citalopram
Clomipramine
-       N-Desmethyl-Clomipramine (Metabolite)
Doxepin
-       Nordoxepin (Metabolite)
Duloxetine
Fluoxetine
-       Norfluoxetine (Metabolite)
Imipramine
-       Desipramine (Metabolite)
Maprotiline
Milnacipran
Paroxetine
Sertraline
-       Norsertraline (Metabolite)
Trazadone
Trimipramine
-       N-Desmethyl-Trimipramine (Metabolite)
Venlafaxine

 

10
10
10
10
25
10
10
100
10
10
10
10
10
10
10
10
10
10
10
25
10
10
10
10

 

1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000

Antihistamines
Dextromethorphan
Diphenhydramine
Hydroxyzine

 

10
10
10

 

1000
1000
1000

Antipsychotics
Chlorpromazine
-       Promazine (Metabolite)
Modafinil
Olanzapine
Quetiapine

 

10
10
100
25
10

 

1000
1000
1000
1000
1000

Barbiturates
Butabarbital
Butalbital
Pentobarbital
Secobarbital
Phenobarbital

 

50
50

50
50

 

5000
5000
5000
5000
5000

Benzodiazepines
7-Aminonitrazepam
Alpha-Hydroxytriazolam
Alprazolam
-       Alpha-Hydroxyalprazolam (Metabolite)
Chlordiazepoxide
-       Norchlordiazepoxide (Metabolite)
Clonazepam
-       7-Aminoclonazepam (Metabolite)
Diazepam
-       Temazepam (Metabolite)
-       Nordiazepam (Metabolite)
-       Oxazepam (Metabolite)
Estazolam
Flunitrazepam
-       7-Aminoflunitrazepam (Metabolite)
Flurazepam
-       2-Hydroxethylfurazepam (Metabolite)
Lorazepam
Midazolam
-       Alpha-Hydroxymidazolam (Metabolite)

 

10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
25
10
10
10

 

1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000

Cannabinoids
-       THC-COOH (Metabolite)
-       11-OH-THC (Metabolite)
-       THC-COOH-glucuronide (Metabolite)

 

2.5
10
10

 

1000
1000
1000

Ethanol
Ethyl glucuronide
Ethyl sulfate

 

250
250

 

5000
5000

Illicit
3-Trifluoromethylphenylpiperazine
6-Monoaceytlmorphine (6-MAM)
Benzylpiperazine
Cocaine
-       Benzoylecgonine (Metabolite)
LSD
-       2-oxo-3-OH-LSD (Metabolite)
Methamphetamine
Methylenedioxymethamphetamine
Methylenedioxypyrovalerone
Phencyclidine (PCP)
Psilocin

 
10
10
50
2.5
10
10
25
50
50
50
10
10

 

1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000

Miscellaneous
Guanfacine
Mitragynine

 

10
10

 

1000
1000

Muscle Relaxants
Baclofen
Carisoprodol
-       Meprobamate (Metabolite)
Cyclobenzaprine
-       N-Desmethyl-Cyclobenzaprine (Metabolite)

 

10
10
10
10
10

 

1000
1000
1000
1000
1000

Nicotine
Cotinine

 

100

 

1000

Opiates
Alfentanil
Buprenorphine
-       Norbuprenorphine (Metabolite)
Butorphanol Tartrate
Codeine
-       NorCodeine (Metabolite)
Fentanyl
-       Despropionylfentanyl (Metabolite)
Norfentanyl
Hydrocodone
-       Norhydrocodone (Metabolite)
-       Hydromorphone (Metabolite)
-       Dihydrocodeine (Metabolite)
Meperidine
-       Normeperidine (Metabolite)
Methadone
-       EDDP (Metabolite)
Morphine
Nalbuphine
Naloxone
Naltrexone
-       Naltrexol (Metabolite)
Pentazocine
Propoxyphene
-       Norpropoxyphene (Metabolite)
Sufentanil

 

10
5
10
10
10
10
10
10
10
10
10
50
10
25
10
10
10
10
10
25
10
10
10
10
10
10

 

1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000

Oxycodone
Oxycodone
-       Noroxycodone (Metabolite)
-       Oxymorphone (Metabolite)

 

10
10
10

 

1000
1000
1000

Stimulants
Amphetamine
-       Norephedrine (Metabolite)
Mephedrone
Methylphenidate
-       Ritalinic acid (Metabolite)
Phentermine
Pseudoephedrine

 

50
10
25
10
10
25
10

 

1000
1000
1000
1000
1000
1000
1000

Z-Drugs
Zaleplon
Zolpidem
Zopiclone

 

10
10
10

 

1000
1000
1000

 

Table 4: Internal Standard Composition


Internal Standard Concentrations

1-3Chlorophenylpiperazine-D8

6-Mam-D6

7-aminoclonazepam-D4

Amitriptyline-D3

Amphetamine-D8

Aripiprazole-D8

Benzoylecgonine-D8

Buprenorphine-D4

Cocaine-D3

Codeine-D6

EDDP-D3

Fentanyl-D5

Fluoxetine-D5

Hydrocodone-D6

Hydromorphone-D6

Lamotrigine-13C-15N4

Meprobamate-D7

Methadone-D3

Methamphetamine-D11

Methylphenidate-D9

Morphine-D3

Naltrexol-D3

Norbuprenorphine-D3

Norpropoxyphene-D5

Nortriptyline-D3

Oxycodone-D6

Phencyclidine-D5

Tramadol-13C-D3

Venlafaxine-D6

Zopiclone-D4

Anesthesiology (SOM)

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