Application Note - Grupo Biomaster

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Application Note Clinical Research Authors Andre Szczesniewski 1 and Carrie J. Adler 2 1 Agilent Technologies, Inc. Wood Dale, IL 2 Agilent Technologies, Inc. Santa Clara, CA Abstract Most of the compounds in large drug panels are analyzed using positive ionization mode. However, barbiturates and 11-nor-9-carboxy-Δ 9 -THC (THC-A) perform better using electrospray ionization (ESI) in negative mode with the mobile phase pH favorable for negative ionization. This work developed a fast analytical method combining eight barbiturates and THC-A in a single analysis using alternating column regeneration (ACR) to increase sample throughput. Moving the analytes preferring negative ionization into a separate test increases the analytical sensitivity of the compounds, allowing for greater research capabilities. The simple sample preparation techniques used provided rapid analysis, good analytical sensitivity, and quantitation over a wide dynamic range. This analysis used an Agilent 6470 triple quadrupole mass spectrometer with Agilent Jet Stream technology in ESI negative ionization mode and an Agilent Infinity II 1290 UHPLC system. A second pump and 2-position 10-port switching valve were added to facilitate use of the ACR. A 100 µL aliquot of human urine was used for the analysis of barbiturates and THC-A. Chromatographic separation of the analytes was achieved in less than 3 minutes using a gradient method composed of a H 2 O:acetonitrile mixture with 5 mM ammonium acetate and an Agilent Polaris C18-Ether, 100 × 2.0 mm, 3.0 µm column. Quantitative analysis was performed using multiple reaction monitoring (MRM) transition pairs for each analyte and an internal standard in the negative ionization mode. The isobaric pair, amobarbital and pentobarbital, were not separated under these chromatographic conditions. Good linearity and reproducibility were obtained for the concentration range from 5 to 1,000 ng/mL with a coefficient of determination >0.995 for all analytes. Excellent reproducibility was observed for all analytes (CV <15 %). A fast, specific, and accurate quantitative LC/MS/MS analytical method was developed and verified for the simultaneous measurement of barbiturates and THC-A in urine. A fast analytical LC/MS/MS method for the simultaneous analysis of barbiturates and 11-nor-9-carboxy-Δ 9 - tetrahydrocannabinol (THC-A) in urine Using ESI negative ionization mode and alternating column regeneration

Transcript of Application Note - Grupo Biomaster

Page 1: Application Note - Grupo Biomaster

Application Note

Clinical Research

AuthorsAndre Szczesniewski1 and Carrie J. Adler2

1 Agilent Technologies, Inc. Wood Dale, IL

2 Agilent Technologies, Inc.Santa Clara, CA

AbstractMost of the compounds in large drug panels are analyzed using positive ionization mode. However, barbiturates and 11-nor-9-carboxy-Δ9-THC (THC-A) perform better using electrospray ionization (ESI) in negative mode with the mobile phase pH favorable for negative ionization. This work developed a fast analytical method combining eight barbiturates and THC-A in a single analysis using alternating column regeneration (ACR) to increase sample throughput. Moving the analytes preferring negative ionization into a separate test increases the analytical sensitivity of the compounds, allowing for greater research capabilities. The simple sample preparation techniques used provided rapid analysis, good analytical sensitivity, and quantitation over a wide dynamic range.

This analysis used an Agilent 6470 triple quadrupole mass spectrometer with Agilent Jet Stream technology in ESI negative ionization mode and an Agilent Infinity II 1290 UHPLC system. A second pump and 2-position 10-port switching valve were added to facilitate use of the ACR. A 100 µL aliquot of human urine was used for the analysis of barbiturates and THC-A. Chromatographic separation of the analytes was achieved in less than 3 minutes using a gradient method composed of a H2O:acetonitrile mixture with 5 mM ammonium acetate and an Agilent Polaris C18-Ether, 100 × 2.0 mm, 3.0 µm column. Quantitative analysis was performed using multiple reaction monitoring (MRM) transition pairs for each analyte and an internal standard in the negative ionization mode. The isobaric pair, amobarbital and pentobarbital, were not separated under these chromatographic conditions. Good linearity and reproducibility were obtained for the concentration range from 5 to 1,000 ng/mL with a coefficient of determination >0.995 for all analytes. Excellent reproducibility was observed for all analytes (CV <15 %). A fast, specific, and accurate quantitative LC/MS/MS analytical method was developed and verified for the simultaneous measurement of barbiturates and THC-A in urine.

A fast analytical LC/MS/MS method for the simultaneous analysis of barbiturates and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-A) in urine

Using ESI negative ionization mode and alternating column regeneration

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IntroductionCompounds in large drug panels are analyzed using positive ionization mode. However, barbiturates and 11-nor-9-carboxy-Δ9-THC (THC-A) perform better in negative ionization mode using a separate assay with the mobile phase pH favorable for negative ionization. Included in the analysis were eight barbiturates and THC-A (Figure 1). barbiturates included: amobarbital, butabarbital, butalbital, methohexital, pentobarbital, phenobarbital, hexobarbital, and secobarbital. This work developed a fast analytical method combining barbiturates and THC-A in a single analysis using alternating column regeneration (ACR) to increase sample throughput. This analytical method used the ability of LC/MS/MS to detect compounds over a wide range of concentrations simultaneously. The calibration concentrations ranged from 0.1 ng/mL to 5,000 ng/mL. The standard curve preparation was generated using matrix-matched standards, diluting 1:10 and injecting into the LC/MS/MS system. The methodology was developed on an Agilent 1290 Infinity II UHPLC and an Agilent 6470 LC/TQ mass spectrometer with a 5-minute analytical gradient. ACR reduced the analysis time by 26 %, to 3.7 minutes injection to injection.

Experimental

LC Configuration and parameters

Configuration

Pump Two Agilent 1290 Infinity II binary pumps (p/n G7120A)

Multisampler Agilent 1290 Infinity II multisampler (p/n G7167A)

Column compartment Agilent 1290 Infinity thermostatted column compartment with a 2-position 10 port valve (p/n G7116B)

Needle wash 50:20:20:10 IPA:MeOH:ACN:H2O

Autosampler temperature 10 °C

Injection volume 10 µL

Analytical column 2 Agilent Poroshell 120 EC-C18, 2.1 × 100 mm, 1.9 µm, LC columns (p/n 695775-902)

Column temperature 55 °C

Mobile phase A 5 mM Ammonium acetate in water

Mobile phase B Acetonitrile

Flow rate 0.35 mL/min

Gradient

Eluent pump

Time (min) %B 0.00 35 1.60 45 1.61 98 3.00 98 3.01 45 3.59 45

Regeneration pump

Time (min) %B 0.00 98 1.40 98 1.50 35

Stop time Eluent pump: 3.65 minutes Regeneration pump: no limit

Valve position0.00 minutes Current position

3.59 minutes Next position

Figure 1. Analyte structures.

Amobarbital

HH

O

OO

N N

Butalbital

HH

O

OO

N N

Methohexital

HH

O

OO

N N

Pentobarbital

H

O

OO

N N

Phenobarbital

HH

O

OO

N N

Secobarbital

HH

O

OO

N N

Butabarbital

HH

O

OO

N N

Hexobarbital

H

OO

O

N

N

CH3

H3C

11-nor-9-Carboxy-∆9-THC

O

O

OH

OH

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Chemicals and reagentsHuman urine used for matrix-matched calibrators was purchased from Golden West Biologicals, Inc, (Temecula, CA). Standards and internal standards were bought from Cerillant Corporation (Round Rock, TX). Sample preparation and LC solvents were acquired from Honeywell (Muskegon, MI).

Sample preparationStandards were spiked into drug-free human urine solution (10 %). The calibration curve was created by serial dilution following a pattern of 1:2:2:2.5. Concentrations ranged from 0.5 to 5,000 ng/mL. Internal standards were added to a final concentration of 200 ng/mL. Then, 10 μL were injected onto the LC/MS system.

Data analysisData acquisition was performed using Agilent MassHunter Acquisition Software (B.08.00). MS/MS transitions were obtained using Agilent MassHunter Acquisition optimizer software to determine optimal parent and product ions, fragmentor voltages, and collision energies. Data were analyzed using Agilent MassHunter Quantitative Analysis Software (B.08.00) and Qualitative Analysis Software (B.07.00).

Figure 2. Alternating column regeneration (ACR) valve configuration.

Waste

Column 2

Column 1

V1

Detector

12 9

3 8

4 7

5 6

10

Regenerationpump

AutosamplerEluent pump

Valve (V1) position 1

Valve (V1) position 2

Waste

Column 2

Column 1

V1

Detector

12 9

3 8

4 7

5 6

10

Regenerationpump

AutosamplerEluent pump

Figure 3. Graphical timeline for ACR analysis.

Eluent pump column 1

Regeneration pump

column 1

Overlappedinjection

ACR Analysis cycle time = 3.65 minutes

Gradient run

Column wash 1.4 minutes Column equilibration

2.2 minutes

%B

%B

RinseV1

0.6 minutes

Draw andinject

0.85 minutes

3 minutes

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Results and discussion

ChromatographyThe main emphasis of this work was increased throughput. Therefore, under these chromatographic conditions, the isobars amobarbital and pentobarbital did not separate, and are reported as a single peak (Figure 4). If separation between amobarbital and pentobarbital is required, adjust the gradient and the run time to achieve baseline separation between those isobars, as shown in Figure 6.

MS/MS Compound information for analytes and internal standards

Compound ISTD?Precursor ion

(m/z)Product ion

(m/z) RT (min)Fragmentor

(V) Collision energy

(V)

Amo/Pentobarbital 225.1 182 1.78 105 12

Amo/Pentobarbital 225.1 42 1.78 105 24

AmoPentobarbital-D5 230.1 42 1.78 105 24

Butabarbital 211.1 168 1.32 165 12

Butabarbital 211.1 42 1.32 165 40

Butalbital 223.1 180 1.45 165 8

Butalbital 223.1 42 1.45 165 36

Butalbital-D5 228.1 42 1.45 165 36

Hexobarbital 235.1 42 1.85 85 20

Methohexital 261.1 42 2.66 65 20

Phenobarbital 231.1 188 1.17 140 8

Phenobarbital 231.1 42 1.17 140 36

Phenobarbital-D5 236.1 42 1.17 140 36

Secobarbital 237.1 194 2.02 170 12

Secobarbital 237.1 42 2.02 170 36

Secobarbital-D5 242.1 42 2.02 170 36

THC-A 343.2 299.2 2.88 125 24

THC-A 343.2 245 2.88 125 36

THC-A-D9 352.2 254.1 2.88 125 32

LC/TQ Mass spectrometer configuration and parameters

Configuration

Instrument Agilent 6470 triple quadrupole mass spectrometer with Agilent Jet Stream

MS/MS mode MRM

Ionization mode Negative

Drying gas temperature 150 °C

Drying gas flow 11 L/min

Nebulizer pressure 30 psi

Sheath gas temperature 350 °C

Sheath gas flow 11 L/min

Nozzle voltage 2,000 V

Capillary voltage 6,000 V

Delta EMV 800 V

Q1/Q2 resolution Unit/Unit

Dwell time 50 ms

Cell accelerator voltage 4 V

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Figure 4. dMRM Chromatogram showing elution of the nine compounds at 500 ng/mL.

1.0 1.1 1.2

Phenobarbital

Hexobarbital

Secobarbital Methohexital

THC-A

Butabarbital

ButalbitalAmo/pentobarbital

1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.00

0.20.40.60.81.01.21.41.61.82.02.22.42.6

×105

Acquisition time (min)

Coun

ts

Figure 5. Chromatograms at lower limit of quantitation (LLOQ).

Phenobarbital5 ng/mL 5 pg OC*

1,106.16

Hexobarbital Secobarbital Methohexital THC-A

Butabarbital Butalbital Amo/pentobarbital×102

Acquisition time (min)

Coun

ts

1.0 1.1 1.2 1.301234567

0.5 ng/mL 500 fg OC

3,409.85 888.02

×103

Acquisition time (min)

Coun

ts

1.7 1.8 1.9 2.00

0.4

0.8

1.2

1.6

2.0

2.45 ng/mL 5 pg OC

×102

Acquisition time (min)

Coun

ts

1.9 2.0 2.1 2.2

1

234567

604.16

5 ng/mL 5 pg OC

×102

Acquisition time (min)

Coun

ts

2.5 2.6 2.7 2.8

1

2

3

4

5

6

4,093

50 pg/mL 50 fg OC

Acquisition time (min)

Coun

ts

2.5 2.6 2.7 2.8

0.5 ng/mL 500 fg OC

1,651.10

×102

Acquisition time (min)

Coun

ts

1.6 1.7 1.8 1.90123456789

5 ng/mL 5 pg OC987.33

×102

Acquisition time (min)

Coun

ts

1.2 1.3 1.4 1.50

1

2

3

4

5

6 5 ng/mL 5 pg OC

987.80

×102

Acquisition time (min)

Coun

ts

1.3 1.4 1.5 1.6 1.7 1.80

1

2

3

4

5

6

×103

0

0.4

0.8

1.2

1.6

2.0

2.4

Figure 6. Chromatogram with amobarbital and pentobarbital baseline separation.

0.1 0.3 0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9 2.1 2.3 2.5 2.7 2.9 3.1 3.3 3.5 3.7 3.9 4.1 4.3 4.5 4.7 4.9 5.1 5.3 5.5 5.7 5.9

5.91

5.14

4.354.19

2.932.462.04

6.1 6.3

Phenobarbital

Secobarbital

THC-A

ButabarbitalButalbital

AmobarbitalPentobarbital

00.20.40.60.81.01.21.4

×105

Acquisition time (min)

Coun

ts

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Calibration curvesAll calibration curves were linear, and a 1/x weighting factor was used. Figure 7 shows examples of calibration curves, and Table 1 lists curve fit correlations (R2). For better visual presentation, amo/pentobarbital and THC-A are shown with logarithmic scales so that all calibration points can be displayed.

×106

×106

×106

×106

Concentration (ng/mL)0 500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 4,500 5,000

Concentration (ng/mL)0 500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 4,500 5,000

Concentration (ng/mL)

Concentration (ng/mL)

0 500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 4,500 5,000

Resp

onse

s

0

0.5

1.0

1.5

2.0

2.5

3.0 y = 648.971186 * x – 23802.955100R2 = 0.9988

Resp

onse

s

-0.10

0.10.20.30.40.50.60.70.80.91.01.11.2

y = 247.235105 * x – 3570.617722

R2 = 0.9999

Resp

onse

s

0

0.5

1.0

1.5

2.0

2.5

3.0 y = 648.971186 * x – 23802.955100

R2 = 0.9988

Phenobarbital

Butabarbital

Amo/Pentobarbital

0 50 100 150 200 250 300 350 400 450 500

Resp

onse

s

00.51.01.52.02.53.03.5 y = 8035.252294 * x + 6009.393127

R2 = 0.9999

THC-A

Figure 6. Example calibration curves.

Table 1. Linear curve correlation coefficients.

Name Transition R2

Amo/Pentobarbital 225.1 & 42.0 0.9963

Butabarbital 211.1 & 42.0 0.9980

Butalbital 223.1 & 42.0 0.9982

Hexobarbital 235.1 & 42.0 0.9928

Methohexital 261.1 & 42.0 0.9916

Phenobarbital 231.1 & 42.0 0.9968

Secobarbital 237.1 & 42.0 0.9974

THC-A 343.2 & 299.2 0.9992

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Quantitation resultsTable 2 shows all quantitation results. This was a 10-point calibration curve ranging from 5 ng/mL to 5,000 ng/mL for all compounds except amo/pentobarbital and THC-A, which ranged from 0.5 ng/mL to 500 ng/mL. All compounds were analyzed down to 1 ng/mL (0.1 ng/mL for amo/pentobarbital and THC-A), and showed a signal-to-noise ratio of 5 or better.

LevelConc.

(ng/mL)

Hexobarbital results Secobarbital results Methohexital results THC-A results

RT Final conc. Resp. RT Final conc. Resp. RT Final conc. Resp. RT Final conc. Resp.

1 2.5/0.25 1.86 3.81 1777 2.03 3.73 365 2.65 2.51 391 2.87 0.21 2471

2 5/0.5 1.85 6.11 3410 2.03 6.02 885 2.65 5.85 604 2.87 0.50 4773

3 10/1 1.85 9.35 7489 2.03 8.98 1559 2.65 9.60 1554 2.87 0.98 8687

4 25/2.5 1.86 19.74 20576 2.04 22.80 4702 2.65 21.59 4594 2.87 2.81 23554

5 50/5 1.86 34.93 39720 2.03 36.86 7898 2.65 33.59 7637 2.87 4.80 39706

6 100/10 1.86 71.48 85748 2.03 79.09 17502 2.65 69.44 16727 2.87 9.87 80784

7 250/25 1.86 230.17 285638 2.03 239.81 54046 2.65 223.89 55890 2.87 27.89 226961

8 500/50 1.85 409.39 511383 2.03 464.13 105055 2.65 399.36 100383 2.87 48.75 396146

9 1000/100 1.85 931.90 1169527 2.03 1076.06 244202 2.65 883.23 223071 2.87 103.10 836973

10 5000/500 1.86 5224.62 6576592 2.03 5005.01 1137604 2.65 5290.95 1340690 2.87 495.35 4018515

Table 2. Quantitation results.

LevelConc.

(ng/mL)

Phenobarbital results Butabarbital results Butalbital results Amo/Pentobarbital results

RT Final conc. Resp. RT Final conc. Resp. RT Final conc. Resp. RT Final conc. Resp.

1 2.5/0.25 1.19 2.68 326 1.33 3.84 558 1.46 3.44 381 1.78 0.24 1155

2 5/0.5 1.18 5.21 1107 1.32 5.60 987 1.46 6.00 988 1.79 0.59 1971

3 10/1 1.19 8.86 2230 1.33 8.96 1802 1.46 8.99 1696 1.78 0.96 3911

4 25/2.5 1.18 23.92 6875 1.33 23.70 5376 1.46 23.48 5129 1.79 2.18 11737

5 50/5 1.18 40.24 11906 1.33 40.86 9540 1.46 39.96 9035 1.79 3.71 21176

6 100/10 1.18 80.44 24298 1.33 74.78 17770 1.46 78.94 18270 1.79 9.20 62732

7 250/25 1.19 255.89 78389 1.33 254.05 61268 1.46 256.42 60317 1.79 23.49 140534

8 500/50 1.18 454.96 139761 1.33 461.05 111491 1.45 464.14 109530 1.79 42.42 257751

9 1000/100 1.18 994.75 306175 1.33 979.64 237317 1.46 1041.56 246330 1.79 88.96 545678

10 5000/500 1.18 5074.33 1563889 1.33 5090.02 1234614 1.46 5019.58 1188795 1.79 523.99 3240808

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For Research Use Only. Not for use in diagnostic procedures.

This information is subject to change without notice.

© Agilent Technologies, Inc. 2018 Printed in the USA, January 29, 2018 5991-8981EN

ConclusionsThis work combined detection of barbiturates and THC-A into a single analytical method. More emphasis was placed on increasing throughput using alternating column regeneration (ACR). To have a fast analytical method, there was no separation between the isobars amobarbital and pentobarbital, and the initial analysis time for all compounds was 5.0 minutes. The addition of ACR reduced the analysis runtime to 3.7 minutes injection to injection, which translates to a 26 % improvement in throughput. Calibration curves for all compounds were linear, with correlations of 0.99 or better. LLOQs for urine-spiked phenobarbital, butalbital, butabarbital, secobarbital, and methohexital were at least 5.0 ng/mL or better, and for amo/pentobarbital and THC-A the LLOQs were 0.5 ng/mL or better.

Assessing potential interferences for this analytical method of urine matrices from different suppliers prepared for LC/MS analysis through a range of more sample preparation techniques is a project for the future.

References1. Workman, H.; et al. A Combined

Method for the Analysis of Barbiturates and 11-nor-9-carboxy Δ9THC in Urine by LC/MS/MS. SOFT 2011, Poster.

2. Cichelli, J.; Doyle, R. M. LC/MS/MS Analysis of Barbiturates in Urine, Oral Fluid and Blood. MSACL 2015, Poster.