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LC-MS/MS Method Development Challenges for the Analysis of
43 Anxiety Medications and Metabolites
Sharon Lupo, Shun-Hsin Liang, Frances Carroll, Justin Steimling, Susan Steinike, Paul Connolly, Ty Kahler; Restek Corporation
Introduction
The use of liquid chromatography coupled with mass spectrometry (LC-MS/MS)
has become a routine method of analysis in forensic and clinical labs. LCMS/MS provides sensitivity, speed, and specificity when analyzing drugs in
complex biological matrices, such as blood and urine. Anxiety medications,
including benzodiazepines, muscle relaxers, hypnotics, sedatives, z-drugs, and
barbiturates are used to treat a variety of conditions and are often abused in
conjunction with other drugs. The presence of isomers and the need to collect
data in positive and negative ion modes can present the analyst with significant
chromatographic challenges.
In this example, methods were developed for the analysis of 43 common
anxiety medications and their metabolites on Raptor™ LC columns. The final
optimized methods utilize water and methanol or acetonitrile mobile phases
modified with 0.1% formic acid under gradient conditions on Restek Raptor™
Biphenyl and Raptor™ C18 2.7µm, 100 x 2.1mm columns. Both columns were
equipped with EXP® 2.7µm, 5 x 2.1mm guard columns of the equivalent phase.
Methods
The anxiety medications were divided into 6 mixtures containing a total of 43
drugs and metabolites (Table 1). Compounds of the same molecular weight
were distributed into different solutions to facilitate compound optimization
and identification. Following infusion, the stock solutions were diluted in water
and injected into a Shimadzu Nexera UHPLC equipped with a SCIEX API 4500™
MS/MS. Detection was performed using electrospray ionization in positive and
negative ion modes with multiple reaction monitoring (MRM).
Discussion
Each solution was analyzed on the Raptor™ Biphenyl 2.7µm, 100 x 2.1mm
column using a linear scouting gradient program and water, methanol, and
acetonitrile mobile phases each modified as follows:
• Acidic - addition of 0.1% Formic acid
• Neutral - addition of 5 mM Ammonium acetate
• Buffered acidic - addition of 0.1% Formic acid and 5 mM Ammonium formate
Although acetonitrile provided a faster run, methanol showed significant
improvement in peak capacity over the gradient. The inclusion of ammonium
formate in the mobile phase reduced sensitivity over formic acid alone - which
increased overall response for most analytes (Figure 1).
While the majority of drugs in the panel ionize best in positive ion mode, the
barbiturates are weak acids in solution and require negative polarity for
ionization. In addition, the barbiturates amobarbital and pentobarbital are
positional isomers and must be chromatographically separated for accurate
identification. Since these isomers can be extremely difficult to resolve, the
separation was attempted on two different phases: the Raptor™ Biphenyl and
the Raptor™ C18. Using the Raptor™ Biphenyl column, a combined analysis of
anxiety drugs and metabolites, including the barbiturates, was achieved with
polarity switching in 8 minutes (Figure 2). This method resulted in ~40%
resolution between the isomers amobarbital and pentobarbital and requires an
LC-MS/MS with sufficient polarity switching speed to accomplish the assay. It
was discovered that near baseline resolution (~95%) of the barbiturate isomers
could be achieved by analyzing the barbiturates separately on the Raptor™ C18
column (Figure 3), while the remaining anxiety drugs and metabolites could be
quickly analyzed on the Raptor™ Biphenyl column (Figure 4). This simpler
approach improves the resolution of barbiturate isomers and is suitable for
mass spectrometers that lack the speed required for combined analysis. The
instrument conditions for all three analyses can be found in Table 2.
Conclusion
 For the combined analysis of anti-anxiety drugs and barbiturates in a single 8
minute run with partial resolution between the barbiturate isomers use the
Raptor™ Biphenyl column.
 For improved resolution of the isomers, amobarbital and pentobarbital, use
the Raptor™ C18 column for a fast 6 minute barbiturates analysis and
analyze the anti-anxiety drugs and metabolites separately on the Raptor™
Biphenyl column in 5.5 minutes.
PATENTS & TRADEMARKS
Restek® patents and trademarks are the property of Restek Corporation. (See www.restek.com/Patents-Trademarks for full list.) Other trademarks appearing in
Restek® literature or on its website are the property of their respective owners. The Restek® registered trademarks used here are registered in the United States
and may also be registered in other countries.
Results
Table 1: Optimized Transitions for 43 Anxiety Medications and Metabolites
Analyte
7-aminonitrazepam
Norketamine
Zolpidem carboxylic acid
Methaqualone
Zopiclone
Meprobamate
7-aminoclonazepam
Flunitrazepam
Diphenhydramine
Flurazepam
α-Hydroxyalprazolam
7-aminoflunitrazepam
Chlordiazepoxide
Midazolam
Cyclobenzaprine
Carisoprodol
Alpha-hydroxymidazolam
n-Desmethylflunitrazepam
Clonazepam
Desalkylflurazepam
α-Hydroxytriazolam
α-Hydroxyethylflurazepam
Precursor Product
Ion
Ion
252.2
121.1
224.1
125.0
338.2
265.1
251.1
132.2
389.2
245.0
219.1
158.2
286.1
121.2
314.2
267.9
256.1
167.0
388.2
315.2
325.1
297.0
284.1
135.0
300.1
227.0
326.2
291.1
276.2
215.0
261.1
176.0
342.2
324.1
300.1
254.1
316.1
270.0
289.0
140.1
359.1
330.9
333.1
211.1
Product
Precursor Product
Ion
Analyte
Ion
Ion
94.2
Zolpidem
308.2
235.2
89.1
Nordiazepam
271.0
139.9
219.0
Phenazepam
349.1
206.2
91.1
Zaleplon
306.1
264.0
217.2
Triazolam
343.1
307.9
97.0
386.3
122.1
Buspirone
250.1
Estazolam
295.1
266.9
239.1
Temazepam
301.1
255.1
152.1
Alprazolam
309.1
280.9
183.0
Diazepam
285.1
153.9
216.2
Prazepam
325.2
271.0
227.1 Phenobarbital 230.9
187.8
282.0
Butalbital
223.0
180.0
248.9
Amobarbital
225.0
182.0
189.0
Pentobarbital
225.0
182.0
62.0
Secobarbital
237.0
193.9
202.9 Metaloxalone 222.1
161.1
198.1
Lorazepam
321.1
275.0
214.1
Oxazepam
287.1
241.2
104.0
Nitrazepam
282.1
235.9
175.9
238.1
125.1
Ketamine
109.0
Product
Ion
218.9
208.0
179.0
236.0
315.0
95.0
205.1
282.9
204.9
192.9
139.9
85.0
84.9
84.8
84.9
84.8
77.1
229.0
268.8
180.1
89.1
Table 2: Optimized Instrument Conditions
Barbiturates +
Anxiety Medications
Barbiturates Only
Anxiety Medications
Only
Raptor™ Biphenyl
(cat# 9309A12)
Raptor™ C18
(cat# 9304A12)
Raptor™ Biphenyl
(cat# 9309A12)
Raptor™ Biphenyl
(cat# 9309A0252)
Raptor™ C18
(cat# 9304A0252)
Raptor™ Biphenyl
(cat# 9309A0252)
Mobile Phase A:
Water +
0.1% formic acid
Water +
0.1% formic acid
Water +
0.1% formic acid
Mobile Phase B:
Methanol +
0.1% formic acid
Acetonitrile +
0.1% formic acid
Methanol +
0.1% formic acid
Flow (mL/min):
0.6
0.6
0.6
Method:
Column:
(2.7 µm, 100 x 2.1 mm)
EXP® Guard Column:
(2.7 µm, 5 x 2.1 mm)
Gradient:
Column Temp.:
Injection Volume:
Time
%B
Time
%B
Time
%B
0.00
35
0.00
20
0.00
30
3.50
70
4.00
28
1.50
80
6.00
100
4.01
20
3.00
95
6.01
35
6.00
20
3.50
95
8.00
35
3.51
30
5.50
30
30 degrees C
50 degrees C
30 degrees C
2 µL of 50-500 ng/mL
standard in water
5 µL of 500 ng/mL
standard in water
5 µL of 10 ng/mL
standard in water
+/-
-
+
Polarity:
Figure 3: Optimized Barbiturate Analysis on the Raptor™ C18.
1.
2.
3.
4.
5.
Phenobarbital
Butalbital
Pentobarbital
Amobarbital
Secobarbital
Chlordiazepoxide
Mobile Phase A: Water + additive; Mobile Phase B: Methanol + additive; Flow: 0.4 mL/min;
Gradient (Time) %B: (0 min) 10% B, (9 min) 100% B, (10 min) 100% B, re-equilibrate
Figure 2: Combined Polarity Switching Analysis – Barbiturates and Anxiety
Medications
Figure 4: Optimized Anxiety Medication Analysis on the Raptor™ Biphenyl.
38 Anxiety Medications & Metabolites
2
4
5
1
Figure 1: Effects of Mobile Phase Additives on Retention and Max. Response
3

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