26 February / March 2017
Table 1. Analyte list, retention times and MS parameters for benzodiazepines and metabolites analysed in this application.
Compound
1 2 3
N-desmethyl Zopiclone
Zopiclone Zolpidem
Flurazepam RT
1.07 1.13 1.62
4 7-aminoclonazepam 1.92 5
2.32
6 7-aminoflunitrazepam 2.36 7 8 9
Chlordiazepoxide Midazolam
10 11 12 13 14 15 16 17 18 19 20 21
1
α-OH-midazolam α-OH-triazolam -OH-alprazolam Oxazepam1 Nitrazepam Lorazepam Clonazepam Alprazolam
Nordiazepam Flunitrazepam Temazepam Triazolam Diazepam
2.35 2.53 2.91 3.78 3.77 3.84 3.87 4.01 4.10 4.35 4.36 4.41 4.45 4.47 5.14
M+H+
375.1 389.1 308.1 286.1 388.2 284.1 300.0 326.0 342.0 359.0 325.1 289.0 282.1 321.0 316.0 309.1 271.0 314.1 301.1 343.0 285.1
productions
245.0 331.0
245.0 111.9
235.1 92.0
121.0 222.1
315.1 100.0
135.0 226.9
227.0 283.0
291.0 222.9
203.0 168.0
176.0 140.8
297.1 243.1
103.9 243.0
180.1 236.0
277.0 229.0
214.1 241.1
205.0 281.1
140.0 165.0
239.2 268.1
177.0 255.1
308.0 239.0
154.0 193.1
MRM voltage
6 6
8 8
34 34
50 50
40 40
34 34
34 34
16 16
2 2
28 28
50 50
50 50
50 50
50 50
54 54
50 50
50 50
50 50
36 50
28 28
50 50
Cone
Collision energy
14 8
12 58
32 52
26 30
26 28
26 22
20 12
36 24
24 40
24 38
25 30
30 20
36 20
20 30
42 40
40 26
30 28
30 25
46 20
24 38
26 30
Oxazepam’s parent ion was set at 289 to avoid interference with Nitrazepam-d5 seen with m/z 287. Recovery
Figure 3 shows the average extraction recoveries of the entire panel of compounds from four separate experiments. All were performed at a concentration of 10 ng/mL. Recoveries ranged from 76-102% with an average of 91%, demonstrating excellent extraction efficiency. The recoveries were consistent as well, with coefficients of variation (%CVs) ranging from 5.2% to 15%, with a mean of 8.6%. The extraction method was changed from a traditional mixed cation exchange (MCX) method for basic compounds. Standard methods use wash steps, 2% aqueous formic acid followed by 100% methanol. The first wash step was modified from aqueous 2% formic acid to 0.02 N HCl to account for the low pKa’s of compounds such as clonazepam, flunitrazepam, and alprazolam and ensure ion-exchange retention on the MCX sorbent. A series of experiments performed during method development revealed that more than 20% methanol in the wash step resulted in loss of the acidic benzodiazepines, such as oxazepam, lorazepam, and temazepam. Thus, the second wash step consisted of 20% methanol, the strongest organic wash possible before breakthrough occurs. These modifications maximised reversed-phase and ion-exchange retention and enabled the highly efficient and most selective extraction of the entire panel of benzodiazepines.
Mean Recovery 120.0% 100.0% 80.0% 60.0% 40.0% 20.0% 0.0% Quantitative Results
Figure 3. Extraction recovery for the compounds in this application. Values represent the mean of 4 individual extractions. Recoveries ranged from 76%-102.5% with an average recovery of 91%. Direct loading of the sorbent, without conditioning and equilibration had no impact on analyte recovery.
Calibration curves ranged from 0.5 ng/mL through 500 ng/mL for all compounds. All compounds had LOQs of 0.5 ng/mL and upper limits of quantitation (ULOQs) of 500 ng/mL. Quality control samples were prepared at 1.5, 7.5, 75 and 300 ng/mL. A calibration summary is shown in Table 2.
Two key benefits of this method take advantage of the water-wettable SPE sorbent: the ability to directly load without conditioning and equilibration, and the ability to conduct all hydrolysis and pretreatment within the well of the SPE plate. The traditional six-step mixed- mode solid phase extraction (SPE) method was simplified into just four steps. This was accomplished by eliminating the conditioning and equilibration steps. Conducting all of the sample hydrolysis and pretreatment steps within the wells of the 96-well plate eliminates the need to transfer the sample from an incubation vessel to the SPE plate, a step that can be time consuming or error prone. After incubation within the wells of the µElution plate, the samples were simply mixed with 4% H3
PO4
to quench the hydrolysis reaction and ionise the basic benzodiazepines, which were then drawn directly onto the sorbent.
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60
