CLINICAL/FORENSICS ARTICLE

Fast, Sensitive Analysis of Benzodiazepines by LC/MS/MS

Quantify an Order of Magnitude below Typical Methods

By Kristi Sellers, Clinical/Forensic Innovations Chemist

Achieve full chromatographic separation of compounds with shared precursor ions.
Increase accuracy with improved desolvation efficiency from a highly organic mobile phase.
Quantify compounds at 10ng/mL or less in urine.

Benzodiazepines are widely prescribed drugs used for treating anxiety and sleep disorders. Since addiction and abuse can occur, efficient screening methods are critical to clinical, forensic, and toxicology laboratories. The liquid chromatography tandem mass spectrometry (LC/MS/MS) method presented here offers several advantages over other techniques: minimal sample preparation, fast analysis times, multiple reaction monitoring transitions for quantification and confirmation, and sensitivity down to 0.10-10ng/mL. This method uses the Allure® PFP Propyl stationary phase, which retains compounds long enough to minimize matrix interferences and chromatographically separate compounds that share the same precursor ion.

Procedure

Samples were prepared by adding 100µL of internal standard solution (1µg/mL D5-Diazepam and D3-Dioxepine) to 100µL urine, diluting with 800µL LC grade water, and centrifuging. The samples were then analyzed by LC/MS/MS. Compound separation was achieved using an Allure® PFP Propyl column and a mobile phase gradient program.

A 3200 QTrap® LC/MS/MS system equipped with a Turbo V™ source with electrospray ionization was used to develop and detect the two MRM transitions (Table 1). For each compound, MRM 1 was used to quantify, and the ratio to MRM 2 was used to confirm.

Cliquid™ Drug Screen & Quant Software was used to process data and generate automatic reporting relevant to forensic guidelines. Limits of quantification were determined and the automated reporting allowed for positive confirmation based on the detected MRM ratios.

Results

By diluting the urine samples ten-fold, matrix effects are reduced (reducing ion suppression) and LOQs between 0.10ng/mL and 10ng/mL can be achieved (Table I). Ion suppression is further reduced by using a retentive column which 1) elutes matrix interferences before the compounds of interest, and 2) allows for better desolvation efficiency due to the ability to use 90% organic in the mobile phase composition. The Allure® PFP Propyl is such a column; it has high retention and selectivity for basic drug compounds, such as benzodiazepines (Figure 1).

The Allure® PFP Propyl stationary phase provides baseline resolution for compounds sharing the same precursor ion, such as nordiazepam and medazepam. The ability to chromatographically separate compounds with similar spectra allows this method to be adapted for single stage MS, however, the LOQ values would be affected. Tandem MS is advantageous since two MRM transitions are collected, allowing quantification and confirmation to be accomplished in a single run, without loss of sensitivity.

Conclusion

The method presented here provides significant advantages over other techniques for benzodiazepine analysis: simple sample preparation, fast analysis time (less than 10 minutes), LOQs of 0.10-10ng/mL in matrix, and quantification and confirmation in a single run.

Further, using the Allure® PFP Propyl column eliminates coelutions of matrix peaks with target analytes and assures full chromatographic resolution of compounds with shared precursor ions.

Figure 1: MRM transitions of 27 benzodiazepines, 3 nonbenzodiazepine hypnotics, and 2 interal standards on the Allure® PFP Propyl column.

Column:

Allure® PFP Propyl

Cat. #:

9169552

Dimensions:

50 x 2.1mm

Particle Size:

5µm

Pore size:

60Å

Sample:

benzodiazepines

Inj.:

20µL

Conc.:

Solvent:

Conditions:

Mobile phase:

A: 0.1% formic acid and 1mM ammonium formate in water

B: 0.1% formic acid and 1mM ammonium formate in acetonitrile

Time(min.)	Flow (µL/min.)	%B
0.0	500	10
10.0	1000	90
15.0	1000	90
15.5	500	10
17.5	500	10

Flow:

see gradient table

Temp.:

40°C

Det.:

Applied Biosystems/MDS Sciex

API 3200™ MS/MS system

Ion Source: Electrospray, positive

IonSpray Voltage: NA

Gas 1: NA

Gas 2: NA

Source Temperature: 500°Cm

LC_PH0463

Data courtesy of Applied Biosystems/MDS Sciex

Table I: MRM transitions, retention times, and LOQ values.
Compound Name	Retention Time (min.)	Precursor Ion (amu)	MRM 1 (amu)	MRM 2 (amu)	DP	CE (MRM 1)	CE (MRM 2)	LOQ (ng/mL)
7-aminonitrazepam	3.2	252.1	121.1	94.0	51	35	53	1.0
7-aminoclonazepam	3.3	286.1	121.0	222.2	46	41	35	0.5
7-aminoflunitrazepam	3.8	284.1	135.1	226.0	51	39	49	0.5
Bromazepam	3.8	316.0/318.0	182.1	182.1	51	45	45	5.0
α-hydroxyalprazolam	4.1	325.1	297.2	204.9	51	31	59	2.0
α-hydroxytriazolam	4.1	359.0	239.2	176.0	61	63	37	5.0
Oxazepam	4.2	287.0	241.1	268.9	41	27	19	10.0
Lorazepam	4.3	321.0/323.1	275.0	277.0	41	31	27	5.0
Estazolam	4.4	295.0	205.0	267.1	51	53	31	2.0
Zaleplon	4.4	306.2	236.3	264.2	56	35	27	0.5
2-hydroxyethylflurazepam	4.5	333.1	211.2	109.0	56	51	41	1.0
Desmethylflunitrazepam	4.5	300.1	254.2	198.2	56	35	51	2.0
Nitrazepam	4.6	282.0	236.1	180.2	71	35	51	2.0
Clonazepam	4.7	316.0	270.2	214.0	56	41	51	2.0
Desalkylflurazepam	4.7	289.1	140.1	226.1	71	41	39	2.0
Temazepam	4.7	301.1/303.1	255.1	257.2	35	30	30	5.0
Triazolam	4.7	343.0	238.9	314.9	61	53	37	1.0
Alprazolam	4.8	309.1	205.1	281.1	56	53	35	1.0
Lormetazepam	4.8	335.0/337.1	289.0	291.1	41	29	29	2.0
Clobazam	4.9	301.1	259.1	224.3	46	29	47	1.0
Flunitrazepam	5.0	314.0	268.1	239.1	56	35	49	1.0
Nordiazepam	5.0	271.1	140.2	164.9	46	37	35	2.0
Zolpiclone	5.4	389.1	244.8	217.0	16	25	41	1.0
D₅-Diazepam	5.4	290.1	198.2	-	55	41	-
Diazepam	5.5	285.0	193.2	154.1	55	41	37	1.0
Chlordiazepoxide	6.0	300.1	227.1	283.2	36	31	21	5.0
Prazepam	6.1	325.1	271.1	140.0	81	31	53	2.0
Zolpidem	7.4	308.1	235.1	236.1	56	39	35	0.2
Midazolam	7.9	326.1	291.3	222.0	56	33	63	0.5
Flurazepam	8.5	388.2	315.1	317.1	36	27	27	0.1
Medazepam	9.0	271.0	91.1	207.3	46	41	39	2.0
D₃-Doxepine	9.1	283.0	107.1	-	41	35	-

Bar color indicates shared precursor ions. Note compounds with shared precursor ions are baseline resolved on the Allure® PFP Propyl column, as shown by retention time comparison.

Data courtesy of Applied Biosystems/MDS Sciex.

Acknowledgement

We sincerely thank Andre Schreiber of Applied Biosystems and Houssain El Aribi and John Gibbons of MDS Sciex for supplying the method and data.

References

Schreiber, Andre PhD, El Arbi, Houssain and Gibbons, John. 2007. A Fast and Sensitive LC/MS/MS Method for the Quantitation and Confirmation of 30 Benzodiazepines and Nonbenzodiazepine Hypnotics in Forensic Urine Samples. Applied Biosystems MDS Sciex.