Enzyme Hydrolysis Workflow for Analyzing Drugs of Abuse in Urine by LC-MS/MS

Formula 1: Fortification Calculation for Hydrolysis Control Samples

Experimental

Master Mix

The following sample preparation uses a master mix containing IMCSzyme RT genetically modified β-glucuronidase (IMCS Irmo, SC). The basic ratio for this master mix is 4 µL of IMCS RT enzyme, 8 µL of IMCS RT buffer, 4.7 µL of water, and 3.3 µL of internal standard (Table I). The total volume of master mix that is prepared should be adjusted to accommodate the number of samples in a batch as each sample is spiked with 20 µL of master mix.

Table I: Internal Standard Concentrations in Urine

Calibrators, Quality Control Samples, and Urine Sample Preparation

Control urine or sample (20 µL) was added to a 1.5 mL microcentrifuge tube along with 20 µL of the premade master mix. Samples were vortexed for 10 seconds and left to incubate at room temperature for 20 minutes. After incubation, 260 µL of diluent (90:10 0.1% formic acid in water:0.1% formic acid in methanol) was added to each sample. The samples were vortexed for 10 seconds and centrifuged for 10 minutes at 3700 rpm. A 100 µL aliquot of supernatant was added to a 200 µL vial insert, and the samples were moved to the LC-MS/MS for analysis. Fortified calibration standards and quality control samples were prepared at the concentrations shown in Table II.

Table II: Analytical Ranges for Drugs of Abuse in Urine

Hydrolysis Control

The hydrolysis control prepared in this method contained four analytes fortified to equal 1000 ng/mL of parent drug when liberated. Morphine-3-β-D-glucuronide; hydromorphone-3-β-D-glucuronide; amitriptyline-N-β-D-glucuronide; and oxazepam glucuronide were spiked into urine using Formula 1. After fortification, 20 µL of the hydrolysis control was added to a 1.5 mL microcentrifuge tube along with 20 µL of the premade master mix. Samples were vortexed for 10 seconds and left to incubate at room temperature for 20 minutes. After incubation, 260 µL of diluent (90:10 0.1% formic acid in water:0.1% formic acid in methanol) was added to each sample. The samples were vortexed for 10 seconds and centrifuged for 10 minutes at 3700 rpm. A 100 µL aliquot of supernatant was added to a 200 µL vial insert, and the samples were moved to the LC-MS/MS for analysis.

Instrument Conditions

Results and Discussion

Optimization of Incubation Time

Different incubation times were tested to determine the optimal time for IMCSzyme RT to perform maximum hydrolysis. This test was performed using a hydrolysis control sample, and incubation times of 10, 15, 20, 25, and 30 minutes were evaluated. An incubation time of 20 minutes produced results closest to the nominal value for all four compounds (Figure 2).

Figure 2: Effect of Hydrolysis Enzyme on Control Samples

Optimization of Diluent Volume

In order to reduce the amount of matrix injected on the column and still achieve the required sensitivity for the method, different diluent volumes (260 µL, 360 µL, 460 µL, and 560 µL) were examined. Overall, 260 µL of diluent was chosen as the best compromise when considering matrix loading, peak shapes, and sensitivity.

Evaluation of Hydrolysis Efficiency

To assess hydrolysis efficiency, three hydrolysis controls were prepared and analyzed over the course of three days (n=9). The control samples showed acceptable results with all analytes falling within ±15% of the expected value for both intraday and interday repeatability studies (Table III). Precision results also passed with %RSD values of <10%.

Table III: Interday Repeatability of the Hydrolysis Control (Average Across Three-Day Study)

Morphine
Sample	Expected (ng/mL)	Average (ng/mL)	% Difference	% RSD
1	1000	1110	11.0	7.6
2	1000	915	8.5	9.3
3	1000	950	5.0	8.9
Hydromorphone
Sample	Expected (ng/mL)	Average (ng/mL)	% Difference	% RSD
1	1000	1040	4.0	2.5
2	1000	1100	10.0	2.4
3	1000	1090	9.0	2.4
Amitriptyline
Sample	Expected (ng/mL)	Average (ng/mL)	% Difference	% RSD
1	1000	1150	15.0	6.9
2	1000	962	3.8	8.3
3	1000	1010	1.0	0.0
Oxazepam
Sample	Expected (ng/mL)	Average (ng/mL)	% Difference	% RSD
1	1000	977	2.3	4.1
2	1000	940	6.0	4.2
3	1000	880	12.0	0.0

Chromatographic Performance

The analysis and separation of 70 drugs of abuse in urine was achieved in a fast, 8-minute cycle time on a Raptor Biphenyl 50 x 2.1 mm, 2.7 µm column by LC-MS/MS as demonstrated in Figure 3.

Figure 3: 70 Drugs of Abuse in Urine Analyzed by LC-MS/MS Following Enzymatic Hydrolysis

LC_CF0806

Peaks

	Peaks	tR (min)	Conc. (ng/mL)	Precursor Ion	Product Ion 1	Product Ion 2	Polarity
1.	Cotinine	0.78	800	177.1	80.0	98.1	+
2.	Morphine	0.85	400	286.2	152.1	165.0	+
3.	Pregabalin	0.88	2000	160.2	142.1	55.0	+
4.	Oxymorphone	0.92	400	302.1	227.2	198.2	+
5.	Hydromorphone	1.08	400	286.2	184.9	156.9	+
6.	Amphetamine	1.13	400	136.2	91.0	65.1	+
7.	Gabapentin	1.18	2000	172.2	154.0	137.1	+
8.	Methamphetamine	1.48	800	150.2	91.1	119.0	+
9.	Phentermine	1.62	800	150.2	91.1	133.1	+
10.	Noroxycodone	1.62	400	302.1	227.0	197.9	+
11.	Naloxone	1.65	160	328.3	310.1	212.3	+
12.	Norhydrocodone	1.73	400	286.1	199.0	128.2	+
13.	<a class="cmpd_link" title="View compound information for O-Desmethyltramadol” title=”View compound information for O-Desmethyltramadol” href=”https://ez.restek.com/compound/view/en/80456-81-1/O-Desmethyltramadol”>O-Desmethyltramadol	1.75	400	250.1	58.0	42.0	+
14.	Codeine	1.80	400	300.2	152.0	165.1	+
15.	MDMA	1.82	400	194.1	163.0	135.1	+
16.	6-Acetylmorphine	1.84	80	328.2	211.0	165.0	+
17.	Oxycodone	1.95	400	316.2	298.0	169.0	+
18.	Naltrexone	2.04	400	342.2	324.0	267.0	+
19.	Hydrocodone	2.06	400	300.2	199.0	128.0	+
20.	<a class="cmpd_link" title="View compound information for O-desmethylvenlafaxine” title=”View compound information for O-desmethylvenlafaxine” href=”https://ez.restek.com/compound/view/en/93413-62-8/O-desmethylvenlafaxine”>O-desmethylvenlafaxine	2.16	400	164.1	58.1	107.0	+
21.	6-β-Naltrexol	2.21	40	344.2	326.1	308.1	+
22.	Lamotrigine	2.24	400	255.9	211.1	145.0	+
23.	Ritalinic acid	2.34	400	220.1	84.1	56.2	+
24.	N-Desmethyltapentadol	2.40	400	208.1	121.2	107.1	+
25.	Norketamine	2.46	400	224.1	125.0	89.1	+
26.	Hydroxybupropion	2.50	400	256.0	130.2	166.0	+
27.	Norfentanyl	2.53	80	233.1	84.1	55.0	+
28.	7-Hydroxyquetiapine	2.71	400	400.2	269.0	208.0	+
29.	Tramadol	2.76	400	264.2	58.0	77.1	+
30.	Xylazine	2.84	400	221.9	90.1	71.9	+
31.	Zolpidem Phenyl-4-carboxylic acid	2.84	400	338.1	265.1	219.0	+
32.	Benzoylecgonine	2.85	200	290.1	168.1	77.1	+
33.	Normeperidine	2.94	400	234.1	160.2	91.0	+
34.	Meprobamate	3.01	400	219.1	158.2	97.0	+
35.	7-Aminoclonazepam	3.07	400	286.1	121.2	250.1	+
36.	Phenobarbital	3.23	800	230.8	187.8	85.0	–
37.	Venlafaxine	3.35	400	278.4	260.4	195.1	+
38.	Mirtazapine	3.39	400	266.1	72.1	195.1	+
39.	Butalbital	3.47	800	222.9	180.0	84.9	–
40.	Norbuprenorphine	3.51	80	414.3	152.2	165.2	+
41.	LSD	3.61	80	324.2	223.1	208.0	+
42.	7-Hydroxymitragynine	3.65	80	415.5	190.1	174.9	+
43.	9-Hydroxyrisperidone	3.77	400	427.2	110.2	207.1	+
44.	Acetyl fentanyl	3.79	40	323.2	188.0	105.0	+
45.	Citalopram	3.94	400	325.1	109.1	262.0	+
46.	Desmethyldoxepin	3.99	400	266.1	107.1	115.0	+
47.	Trazodone	4.10	400	372.3	148.0	260.4	+
48.	Dextromethorphan	4.18	400	272.1	215.1	170.9	+
49.	Haloperidol	4.18	400	377.2	170.9	123.0	+
50.	Fentanyl	4.20	40	337.2	188.0	105.1	+
51.	Norfluoxetine	4.23	400	296.3	134.3	104.9	+
52.	PCP	4.25	200	244.1	86.1	159.1	+
53.	Buprenorphine	4.27	40	468.3	55.1	414.2	+
54.	Carisoprodol	4.43	400	261.1	176.0	62.0	+
55.	EDDP	4.64	400	278.1	234.3	249.2	+
56.	Duloxetine	4.65	400	298.1	154.1	188.2	+
57.	Paroxetine	4.65	400	330.1	192.2	70.1	+
58.	Nortriptyline	4.67	400	264.1	91.1	115.2	+
59.	Cyclobenzaprine	4.67	400	276.2	215.0	189.0	+
60.	Sufentanil	4.70	40	387.2	238.1	111.1	+
61.	Amitriptyline	4.82	400	278.1	91.1	202.1	+
62.	Norsertraline HCl	5.02	400	292.0	275.0	159.0	+
63.	Lorazepam	5.02	400	321.1	229.0	275.0	+
64.	Methadone	5.08	400	310.2	264.9	105.1	+
65.	Oxazepam	5.08	400	287.1	268.8	241.2	+
66.	Dehydro aripiprazole	5.18	400	446.2	285.0	98.1	+
67.	alpha-Hydroxyalprazolam	5.33	400	325.1	297.0	216.2	+
68.	Nordiazepam	5.40	400	271.0	139.9	208.0	+
69.	Temazepam	5.71	400	301.1	255.1	282.9	+
70.	THC-COOH	6.73	200	343.0	298.9	244.8	–

Conditions

Column	Raptor Biphenyl (cat.# 9309A52)
Dimensions:	50 mm x 2.1 mm ID
Particle Size:	2.7 µm
Pore Size:	90 Å
Guard Column:	Raptor Biphenyl EXP guard column cartridge 5 mm, 2.1 mm ID, 2.7 µm (cat.# 9309A0252)
Temp.:	45 °C
Standard/Sample
Diluent:	90:10 Water, 0.1% formic acid:methanol, 0.1% formic acid
Inj. Vol.:	2 µL
Mobile Phase
A:	Water, 0.1% formic acid
B:	Methanol, 0.1% formic acid
	Time (min) Flow (mL/min) %A %B 0.00 0.6 90 10 6.00 0.6 25 75 7.00 0.6 0 100 7.01 0.6 90 10 8.00 0.6 90 10

Detector	SCIEX Triple Quad 4500
Ion Source:	Electrospray
Ion Mode:	ESI+/ESI-
Instrument	Shimadzu Nexera X2
Sample Preparation	20 µL of a calibrator control in urine was added to a 1.5 mL microcentrifuge tube along with 20 µL of a premade enzyme hydrolysis master mix. The sample was vortexed for 10 seconds and left to incubate at room temperature for 20 minutes. After the incubation, 260 µL of the diluent [water, 0.1 % formic acid: methanol, 0.1 % formic acid 90:10 (v:v)] was added. The sample was vortexed for 10 seconds and centrifuged for 10 minutes at 3700 rpm. One hundred microliters was added to a vial insert (cat. #21776) in a 2.0 mL, amber, short-cap vial (cat.# 21142) and capped with a 9 mm short cap (cat.# 24497) and injected on the LC-MS/MS for analysis.

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Resolution of Isobars

Drugs of abuse assays often contain multiple isobars that require chromatographic separation. These analytes must be resolved in order to accurately quantitate each compound because they cannot be distinguished by the MS alone. A target resolution of 1.5 (baseline) or greater is ideal for quantitative work. Resolution can be calculated using Formula 2 where tR is retention time and W is peak width.

Formula 2: Calculation of Resolution

As shown in Table IV, nine groups of isobars were analyzed, and the resolution values within each group were calculated using Formula 2. The Raptor Biphenyl column provided good selectivity and effectively separated the compounds within all nine isobar groups.

Table IV: Isobar Resolution at High QC

Isobar Group	Name	Molecular Weight (g/mol)	Retention Time (min)	Peak Width	Resolution
1	Morphine	285.3	0.87	0.105	1.9
	Hydromorphone		1.11	0.150
	Norhydrocodone		1.75	0.122	8.7
	7-Aminoclonazepam		3.07	0.180
2	Oxymorphone	301.3	0.95	0.12	5.8
	Noroxycodone		1.65	0.12
3	Methamphetamine	149.2	1.41	0.15	1.2*
	Phentermine		1.56	0.11
4	Naloxone	327.3	1.65	0.15	1.5
	6-Acetylmorphine		1.84	0.1
5	Codeine	299.4	1.80	0.113	1.7
	Hydrocodone		2.07	0.201
6	O-desmethylvenlafaxine	263.4	2.11	0.15	3.3
	Tramadol		2.70	0.203
	Mirtazapine		3.39	0.201	7.2
	Nortriptyline		4.66	0.150
7	Lamotrigine	256.1	2.24	0.150	1.6
	Hydroxybupropion	255.7	2.50	0.185
8	Citalopram	324.4	3.98	0.2	6.6
	Alpha-hydroxyalprazolam		5.36	0.22
9	EDDP	278.1	4.64	0.11	1.5
	Amitriptyline		4.81	0.12

*If additional resolution of methamphetamine and phentermine is required, a Raptor Biphenyl 50 x 4.6 mm, 2.7 µm column is recommended.

Accuracy and Precision

Precision and accuracy analyses were performed over the course of three days using three sets each day (n=9). Method accuracy was successfully demonstrated with QC LLOQ, QC Low, QC Med, and QC High results falling within ±15% of the expected values for all analytes. The percent relative standard deviation (%RSD) for intraday and interday testing fell below 9.69%, indicating acceptable method precision.

Linearity

Calibration curves were built using standard over internal standard ratios. Linearity was demonstrated using a 1/x² weighted linear regression, and all analytes showed acceptable R² values of 0.991 or greater. The example calibration curves shown in Figure 4 highlight three analytes that represent early, middle, and late eluting compounds: morphine (0.85 min); benzoylecgonine (2.85 min); and temazepam (5.71 min). The linear ranges varied across the different drugs of abuse and are presented above in Table II.

Figure 4: Calibration Curves for Selected Drugs of Abuse

Column Robustness

Because urine is a relatively dirty matrix, using a guard column is recommended to remove matrix components and protect the analytical column from contamination. Column robustness was tested by running more than 250 matrix injections on the same guard column and analytical column. This evaluation showed good results, with the retention times of the first and last injections having a percent difference of 4.52% or less for all analytes with no observed increase in back pressure. This demonstrates that the performance of both the guard and analytical columns is robust over many injections.

Conclusion

The method developed here provides a quick, effective approach for sample preparation and LC-MS/MS analysis of 70 drugs of abuse in urine. Separation of all drugs, including isobars, was achieved with this rapid and reliable 8-minute method, allowing high-throughput, quantitative analysis at trace levels. This method demonstrated successful precision, accuracy, and linearity for all analytes. It also showed that enzymatic hydrolysis was effective in cleaving the glucuronide from the analyte of interest, allowing lower limits of detection and the ability to report total concentrations.

References

1. K. E. Moeller, et al., Urine drug screening: Practical guide for clinicians. Mayo Clinic Proceedings. 83, (1) (2008) 66-76. https://www.mayoclinicproceedings.org/article/s0025-6196(11)61120-8/fulltext
2. J. Cybulski, Urine analysis: The good, the dad, and the ugly. LCGC International. 12 (2016) (3) 13-16. https://www.chromatographyonline.com/view/urine-analysis-good-bad-and-ugly
3. J. Neifeld, Urine hydrolysis: how did I choose which enzyme to use? Blog. Biotage
   https://www.biotage.com/blog/urine-hydrolysis-how-did-i-choose-which-enzyme-to-use   (Accessed April 17, 2025)
4. K. Skov, et al.,  Exploring enzymatic hydrolysis of urine samples for investigation of drugs associated with drug-facilitated sexual assault. Pharmaceuticals. 17 (2023) (21) https://doi.org/10.3390/ph17010013

This method has been developed for research use only; it is not suitable for use in diagnostic procedures without further evaluation.

Products Mentioned

---

Catalog No. 9309A52

Colonna HPLC Raptor bifenilica, 2,7 µm, 50 x 2,1 mm

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Catalog No. 9309A0252

Cartuccia per precolonna Raptor bifelinica EXP, 2,7 µm, 5 x 2.1 mm, 3 pz.

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Catalog No. 21142

Vial con tappo corto con indicatore graduato, filettatura 9-425, 2,0 mL, 9 mm, 12 x 32 (solo vial), ambrato, 100 pz.

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Catalog No. 24497

Tappi corti a vite, polipropilene, filettatura, setti in PTFE/silicone/PTFE, blu, preassemblati, 2,0 mL, 9 mm, 100 pz.

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Catalog No. 25808

Holder EXP a connessione diretta per cartucce per precolonne EXP, comprende raccordo e ferrule

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Author

• 
  Samantha Herbick

  Samantha Herbick is an applications scientist within the LC Solutions Department at Restek. Her primary focus is on the development of applications in the toxicology and life science markets. She attended Duquesne University where she earned a bachelor’s degree in Biochemistry and a master's degree in forensic science and law. Prior to joining Restek, Samantha worked as a scientist and method developer in a forensic toxicology lab. In this role, she performed analysis on toxicology casework and worked on the development and validation of new and existing assays using LC-MS/MS, GC-MS, and GC-FID.

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CFAN4417

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