Simplify the LC-MS/MS analysis of Alcohol Metabolites, Barbiturates, and Drugs of Abuse by using One Column and Mobile Phase Setup

Efficiency is key when running multiple drug panels and any way to save time or streamline a process can help reduce costs. At Restek we are always looking for ways to simplify workflows for our customers to make their analyses better, faster, and simpler. Pain management labs are often interested in consolidating their materials and methodology. To help simplify the analysis of alcohol metabolites, barbiturates, and drugs of abuse, three different methods were developed for each analyte class using the same column (Force Biphenyl 50 x 3 mm, 3 µm) and mobile phase setup (0.1% formic acid in water and 0.1% formic acid in methanol). The fully porous nature of our Force particles gives greater surface area than its superficially porous counterpart, allowing for greater retention of analytes and separation of critical isobars. The biphenyl stationary phase provides superior selectivity and increased retention of dipolar, unsaturated, or conjugated compounds when compared to a C18 and is further enhanced by the utilization of methanol as the organic modifier. Restek has the only fully porous Biphenyl on the market and is available in our Force line of LC-MS/MS performance columns. In the following examples we are able to demonstrate the advantage of this set up for multiple panels.

The following analyte panel has been compiled from multiple drug classes that are typically analyzed by LC-MS/MS in ESI (+). Each of the compounds in Table 1 is isobaric with another compound on the list and was chosen to demonstrate the unique selectivity of the biphenyl stationary phase.

Analyte	Retention Time (min)	Precursor	Product 1	Product 2
Methamphetamine	2.93	150.3	91.1	119.2
Phentermine	3.07	150.3	91.1	133.2
Norcotinine	1.35	163.1	80.1	118.0
Nicotine	1.39	163.2	130.1	117.1
Anabasine	1.51	163.2	92.1	120.1
Cotinine	2.02	177.2	80.1	98.1
BZP	1.44	177.2	91.1	65.1
MDA	2.98	180.2	163.1	105.2
Methylephedrine	2.68	180.2	162.2	115.2
Levamisole	3.31	205.1	178.1	91.2
Psilocin	2.42	205.2	58.3	160.1
Methylone	3.07	208.1	160.2	132.2
N-Desmethyltapentadol	3.63	208.1	107.2	121.2
MDEA	3.54	208.2	163.2	105.2
Metaxalone	5.61	222.1	161.2	77.1
Tapentadol	3.76	222.2	107.2	121.2
Normeperidine	4.06	234.1	160.2	91.0
Methylphenidate	4.00	234.4	84.2	56.1
O-Desmethyltramadol	3.02	250.1	44.0	232.0
N-Desmethyltramadol	3.98	250.2	44.1	232.1
Methaqualone	6.10	251.1	32.1	91.2
Lacosamide	4.03	251.5	91.2	108.2
Desmethylmirtazapine	4.27	252.1	195.2	209.2
7-aminonitrazepam	3.34	252.2	121.1	94.2
Phenytoin	5.24	253.0	182.2	104.2
Oxcarbazepine	5.22	253.1	180.1	208.1
Hydroxybupropion	3.80	256.0	166.0	130.2
Ketorolac	5.89	256.1	105.2	77.1
Lamotrigine	3.62	256.1	210.9	145.0
Diphenhydramine	4.76	256.2	167.2	152.1
Tolmetin	6.03	258.1	119.2	91.1
Dextrorphan	3.84	258.2	157.1	201.2
Levorphanol	3.84	258.2	157.2	199.2
Protriptyline	5.18	264.1	191.1	165.0
Nortriptyline	5.29	264.1	91.0	115.0
Lisdexamfetamine	1.82	264.2	84.2	91.2
Desmethylvenlafaxine	3.38	264.2	58.2	107.0
Tramadol	3.86	264.2	58.2	77.0
EMDP	5.68	264.2	220.1	235.2
Mirtazapine	4.40	266.1	195.2	107.2
Desmethyldoxepin	4.82	266.1	115.0	107.2
Desomorphine	3.18	272.1	165.0	152.2
Dextromethorphan	5.01	272.2	215.2	171.1
Normorphine	1.56	272.3	165.1	152.0
MDPV	4.29	276.2	126.3	135.2
Cyclobenzaprine	5.26	276.2	215.1	189.0
Molindone	3.81	278.1	100.1	70.1
Maprotiline	5.26	278.1	250.3	191.1
Amitriptyline	5.27	278.2	202.1	91.2
EDDP	5.27	278.2	234.1	249.2
Venlafaxine	5.27	278.4	260.2	121.2
Promazine	5.19	285.1	86.2	58.2
Diazepam	6.74	285.1	193.1	154.2
Psilocybin	2.04	285.3	205.2	58.3
Norcodeine	2.69	286.1	152.1	115.0
7-aminoclonazepam	4.24	286.1	121.2	250.1
Hydromorphone	2.41	286.2	185.1	157.2
Norhydrocodone	3.05	286.2	199.1	128.2
Morphine	2.16	286.2	152.1	165.1
Pentazocine	4.38	286.2	218.2	69.1
Asenapine	5.11	286.2	166.0	229.0
Noroxymorphone	1.80	288.2	270.2	213.2
Dihydromorphine	2.11	288.3	157.1	185.0
Norcocaine	4.29	290.1	168.2	136.2
Benzoylecgonine	4.11	290.2	168.2	77.2
Estazolam	6.44	295.1	267.1	205.1
Trimipramine	5.40	295.2	100.2	58.3
Didesmethyl citalopram	4.73	297.1	109.2	262.1
Eslicarbazepine	5.65	297.2	194.2	237.1
N-desmethylflunitrazepam	5.82	300.1	254.1	198.1
Chlordiazepoxide	5.05	300.1	227.1	282.0
Codeine	3.02	300.2	152.0	165.0
Hydrocodone	3.27	300.2	199.2	128.2
Clobazam	6.28	301.0	259.1	224.1
Temazepam	6.43	301.1	255.2	283.1
Noroxycodone	2.98	302.1	284.2	227.1
Oxymorphone	2.23	302.2	227.1	198.1
Dihydrocodeine	2.98	302.2	199.2	128.0
Morphine-N-oxide	2.31	302.3	285.2	162.1
Ezogabine	4.84	304.1	109.2	230.1
Cocaine	4.20	304.2	182.2	77.1
Sertraline	5.56	306.1	158.9	275.1
M-hydroxy benzoylecgonine	3.59	306.2	168.2	121.1
Zaleplon	6.32	306.2	236.1	264.2
Fluoxetine	4.91	310.1	44.1	148.0
Methadone	5.55	310.2	265.2	105.2
N-desmethylclozapine	4.25	313.0	192.1	270.1
Olanzapine	2.66	313.2	256.1	198.1
Flunitrazepam	6.39	314.2	268.1	239.1
Amoxapine	5.21	314.2	271.1	193.1
THC	7.15	315.2	193.0	123.1
Clomipramine	5.53	315.3	86.2	58.2
Clonazepam	5.97	316.1	270.1	214.1
Oxycodone	3.19	316.2	298.2	169.0
Alpha-Hydroxyalprazolam	6.17	325.2	297.1	216.2
Prazepam	7.04	325.2	271.2	140.1
Citalopram	4.75	325.2	109.2	262.1
Escitalopram	4.75	325.3	109.2	262.1
Midazolam	5.16	326.1	291.2	223.0
Norpropoxyphene	5.08	326.4	252.2	91.0
Loxapine	5.25	328.2	271.1	193.1
6-Acetylmorphine	3.04	328.2	165.1	211.2
Clozapine	4.49	328.2	271.0	193.0
JWH-073	7.54	328.2	127.1	155.1
Naloxone	2.90	328.3	271.1	310.1
Butorphanol	4.47	328.3	310.2	131.0
Paroxetine	5.23	330.2	192.2	70.2
XLR-11	7.02	330.3	125.1	144.1
Tenoxicam	5.32	338.1	121.2	95.0
Zolpidemphenylcarboxylic acid	3.82	338.2	265.1	219.0
Topiramate	4.76	340.0	264.1	224.0
Propoxyphene	5.09	340.3	58.2	266.3
Alpha-hydroxymidazolam	5.48	342.2	324.1	203.1
Naltrexone	3.24	342.2	324.3	267.2
JWH-018	7.64	342.3	155.2	127.1
Alpha-hydroxytriazolam	6.04	359.2	331.1	176.0
Etoricoxib	5.95	359.2	280.1	279.1
Parecoxib	6.32	371.2	234.1	165.0
Thioridazine	6.03	371.3	126.3	98.2
Haloperidol	4.80	377.2	123.1	95.0
Remifentanil	4.13	377.4	113.2	317.2
Sufentanil	5.16	387.3	238.2	111.1
Mesoridazine	5.44	387.3	126.1	98.2
9-hydroxyresiperidone	4.51	427.0	207.2	110.2
Iloperidone	4.51	427.1	207.2	110.2
Paliperidone	4.51	427.4	207.2	110.1
Pimozide	5.43	462.1	109.2	328.2
Morphine-6-glucuronide	2.22	462.3	286.2	201.1

Table 1: Isobars monitored in these experiments by LC-MS/MS ESI (+) using the conditions listed in Figure 1.

Figure 1: 129 drugs and drug metabolites listed in Table 1 analyzed using the outlined conditions in the figure. A) Separation achieved for seven isobaric compounds that share the m/z of 286.

All positive mode compounds were prepared and analyzed by the outlined conditions. The results of the 129 compound positive mode drug and drug metabolite panel can be seen in Figure 1. An example of the powerful selectivity of the phase is demonstrated for compounds sharing the precursor ion of m/z 286 (Figure 1A). Here, 5 opiate compounds, a benzo metabolite, and an antipsychotic are baseline resolved.

Next, a urine sample was hydrolyzed to detect glucuronide forms of drugs and was demonstrated using ICMS rapid hydrolysis buffer® and ICMSzyme®. 100 µL of urine was aliquoted to a 2 mL centrifuge tube and spiked with analytes. 30 µL of ICMSzyme® and 40 µL of ICMS rapid hydrolysis buffer® were added and vortexed. The sample was transferred to a water bath at 45⁰C for 30 minutes. 730 µL of 0.1% formic acid in water was added to quench the reaction. Samples were centrifuged at 4200 rpm for 5 minutes and transferred to a 0.2 µm filter vial and injected onto the LC-MS/MS instrument. The results were compared to a blank and no interferences were observed from hydrolysis.

When applying compounds to matrix, it is important to be able to separate matrix interferences from analytes. In the case of methadone and fluoxetine, not only is it imperative to be able to resolve these isobars from one another, but also to resolve matrix interference from the analytes. In the next example we are able to perform this task using the same conditions outlined in Figure 1 and demonstrate great resolution between interferences and isobars.

Figure 2. Separation of methadone and fluoxetine from urinary matrix interference using conditions outlined in the figure.

In this next example, the same mobile phases and column are also able to analyze barbiturates, THC-COOH, and THCA-A and achieve a partial resolution between amobarbital and pentobarbital using the conditions outlined in Figure 3.

Figure 3: Chromatogram obtained using conditions in the figure for barbiturates, THCA-A, and THC-COOH analyzed in ESI (-) mode.

The partial resolution of amobarbital and pentobarbital allows labs to identify which barbiturate is present in their sample which may eliminate the need for confirmatory testing.

Finally, a method was developed for the analysis of EtG and EtS in urine outlined in Figure 4. Figure 4 shows a chromatogram in urine with a 1:10 dilution in water and utilizes a high organic ramp to facilitate removal of dirty matrix from the column to prevent clogging.

Figure 4: Chromatogram obtained for EtG/EtS in urine using the sample preparation and conditions outlined in the figure.

A panel of 129 drug and drug metabolite isobars in positive mode, negative mode drug and drug metabolites, and alcohol metabolites were all analyzed using the same column and mobile phases. This was achieved using a Force Biphenyl column, which has unique selectivity due to the pi-pi interactions for drugs and drug metabolites when compared to a routine C18 phase. Urinary interferences that are particularly problematic in alcohol metabolite testing were resolved without the use of buffer or additional mobile phases helping to streamline analytical testing processes. The use of an UltraShield PreColumn filter helps prevent buildup on the guard and analytical column, improving their lifetime. Looking to combine your panels? Apply Force!