the quechers approach to determine pharmaceuticals and

The QuEChERS Approach to Determine Pharmaceuticals and Toxins in Whole

Blood

Joan Stevens, Ph.D., Chemistries and Supplies Division, Agilent Technologies, Inc.

3/23/2011

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

IntroductionBlood is the most complex of the biological fluids

Determination of drugs in whole blood is often necessary in forensic analysis because of the difficulty in obtaining serum or plasma

Conventional procedures to analyze drugs in complex matrices like whole blood involve tedious, time consuming, expensive, and complex steps, and possible sample loss and contamination problems are not unusual

QuEChERS is a sample preparation technique that was developed for the extraction of multi-class pesticide residue from fruits and vegetables, in 2003

Although biological fluids may seem vastly different than a piece of fruit they follow parallel paths

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Comparison

Biological Fluids

Complex matrices

Disrupt cell architecture – Lyses, vortexing. organic

solvent, hydrolysisAnalytes with a wide range of polarities

Remove analyte or matrixAnalysis by LC/MS/MS, GC/MS or GC/MS/MS

Fruits and Vegetables

Complex matrices

Disrupt cell architecture– Mincing, vortexing, organic

solvent, hydrolysisAnalytes with a wide range of polarities

Remove analyte or matrixAnalysis by LC/MS/MS, GC/MS or GC/MS/MS

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Basic SPE Procedure for Blood Sample

• Pretreatment of whole blood involves dilution with an acidic solution, phosphate buffer or water/FA

• Sonicate 15 minutes; disrupts cell membranes

• Centrifuge– SPE procedure

• Condition cartridge: methanol, water• Load cartridge: sample

• Wash cartridge: aqueous methanol or buffer % methanol• Elut cartridge: eluent for analysis

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Basic QuEChERS Procedure

15 g homogenized sample, (spike)

Add 15 mL ACN (1% AA), vortex

Add AOAC Extraction Salts, shake

Centrifuge

Transfer 1 or 8 mL to AOAC d-SPE tube, vortex, centrifuge

Transfer to Analysis vial

CN (1

xtrac

entr

OAC

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Advantages of QuEChERS Procedure

• Liquid-liquid type extraction

• Use of acid to interrupt protein binding

• Excess salts to lyses cellular components

• Use of acetonitrile to facilitate protein precipitation

• Use of dispersive SPE to adsorb matrix interferences

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Plossl, et.at., J Chrom A, 1135 (2006), 19-26

•


Disadvantages of QuEChERS Procedure

• Sample and solvent amount required in the method, are not “biological friendly”

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Opposing Sample Preparation Ideas

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Compatible Sample Preparation Techniques

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Purpose Behind the mini-Q Approach

• Offer a more biological friendly approach

• Small sample and volume requirements

• Range of drug compounds, various polarities and pKa

• Evaluation of background interferences

• Extraction and recovery data in acceptable range

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mini-QuEChERS: mini-QProcedure:• Add 2 ceramic homogenizers• 1 or 2 mL whole blood added to a centrifuge tube with screw

cap• Add std soln or IS soln, vortex• Add 2 mL acidified ACN, vortex 30 sec• Add 500 mg of extraction salts, vigorously shake 1 minute,

centrifuge 5000 rpm, 1 min• Transfer 1 mL of extract to d-SPE material in centrifuge tube,

vortex, centrifuge • Add 200 uL to 800 uL water in LC vial, vortex analyze

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Refinement of mini-Q ProcedureParameters evaluated in the mini-Q technique

• Whole blood sample– Most complicated biological matrix

• Addition of acid, type and amount– Effect on partitioning and extraction

• Addition of QuEChERS salts, non-buffered, NaAcetate, or Citrate buffered– Effect on extract, visual inspection

• Dispersive SPE– Matrix cleanup and compound recoveries

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Addition of Acid to Acetonitrile extraction solvent

• Use of acetic acid versus formic acid – Evaluation of a series of acidic acetonitrile solutions in which the percentage

of acid was increased

ACN 1% AA/ACN ACN

Clumping and sticky results

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• Addition of 0.4% formic acid/acetonitrile

EN: citrate buffers AOAC: NaAcetate Non-buffered: NaCl

Lyses of cellular structure, increased particulate nature

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• Evaluation of QuEChERs salts: Partitioning/Extraction– EN: NaCl, Sodium citrate buffers, Mg SO4– AOAC: NaAcetate and MgSO4

– Nonbuffered: NaCl and MgSO4

EN: Citrate salts AOAC: NaAcetate Non-buffered: NaCl

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• Dispersive SPE– Remove matrix interferences: PSA*, C18, GCB, MgSO4– D-SPE: PSA and MgSO4

*PSA: Primary and secondary amine

d-SPE EN: Citrate AOAC: NaAcetate Non-buffered: NaCl EN d-SPE AOAC: d-SPE AOAC: d-SPE AOAC: d-SPE

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Pharmaceuticals Used in StudyCompound CAS number Log P (o/w) pKa Therapeutic Use

Lidocaine 137-58-6 2.4 8.01 Local anesthetic,antiarrhythmic

Tramadol 27203-92-5 2.5 9.41 Analgesic

Amitriptyline 50-48-6 4.92 9.4 Antidepressant

Biperidene 514-65-8 4.0 10.8 Anticholinergic

Oxazepam 604-75-1 2.23 1.7, 11.3 Antianxiety

Lorazepam 846-49-1 2.47 1.3, 11.5 Antidepressant

Chlorpromazine 50-53-3 5.18 9.3 Antipsychotic

Diltiazem 42399-41-7 3.63 7.7 Calcium channel blocker

Naloxone 465-65-6 1.45 7.9 Opioid Receptor Antagonist

Nortriptyline (IS) 72-69-5 5.65 9.7

5 ug/mL stock solution (9 compounds) and IS in methanol

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LC/MS/MS Acquisition DataCompound MRM channels (m/z) Fragmentor (V) CE (V) RT (min) Delta RT

Lidocaine 1) 235.18 > 86.12) 235.18 > 58.1

97 1135

1.37 0.4

Tramadol 1) 264.2 > 58.12) 264.2 > 246.1

97 153

1.20 0.4

Amitriptyline 1) 278.2 > 1172) 278.2 > 105

112 1919

4.25 0.4

Biperidene 1)312.23 > 98.12) 312.23 > 55.1

123 1960

4.23 0.7

Oxazepam 1) 287.06 > 240.92) 287.06 > 268.9

112 197

3.99 0.4

Lorazepam 1) 321.02 > 274.92) 321.02 > 302.9

113 157

4.09 0.4

Chlorpromazine 1) 319.11 > 86.12) 319.11 > 58.1

112 1543

4.63 0.4

Diltiazem 1) 415.17 > 177.92) 415.17 > 149.9

128 1943

3.73 0.4

Naloxone 1) 328.16 > 3102) 328.16 > 212

123 1539

0.82 0.4

Nortriptyline (IS) 1) 264.18 > 2332) 264.18 > 91

97 719

4.17 0.4

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7x10

0.125

0.150.175

0.2

0.2250.25

0.275

0.3

0.325

0.350.375

0.4

0.4250.45

0.475

0.5

0.525

0.550.575

0.6

0.6250.65

0.675

0.7

0.725

0.750.775

0.8

0.8250.85

0.875

0.9

0.925

0.950.975

1

1.025

1.05

1.075

1.11.125

1.15

1.1751.2

1.225

1.25

1.275

1.31.325

1.35

1.375

+ESI TIC Scan Frag=80.0V AOAC_BLANK_110228b.d

1 1

Counts vs. Acquisition Time (min)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10

QuEChERS Extraction of Whole Blood MS Scan mass range 100-800, scan time 20 sec, Fragmentor 80 V, positive polarity. Injection volume 10 μL, 20% B to 75% B over 5.5 minutes. Hold 75% B for 2 minutes.

Poroshell 120 EC-C18, 2.7 μ, 2.1 x 100 mm, PN 695775-902Mobile Phase A: 5 mM Ammonium acetate, pH 5; 20:80 MeOH:WaterB: 5 mM Ammonium Acetate in ACNGT (ºC): 300 GF (l/min): 7Nebulizer (psi): 40SGT (ºC): 400SFG (l/min): 12Capillary (V): 3500NV (V): 500

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3x10

0.1

0.2

0.3

0.40.50.6

0.7

0.80.9

1

1.1

1.21.3

1.4

1.51.6

1.71.8

1.9

2

2.12.2

2.3

2.4

2.5

2.62.72.8

2.93

3.1

3.2

3.3

3.43.5

3.6

3.73.8

3.94

4.1

4.2

4.34.4

4.5

4.6

4.74.84.9

5

5.1+ESI MRM Frag=112.0V [email protected] (278.2000 -> 117.0000) HQC_6.d

1 1

Counts vs. Acquisition Time (min)0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 4.4 4.5 4.6

Nal

oxon

e

Lido

cain

e

Tram

adol

Oxa

zepa

mLo

raze

pam

Bipe

ride

n

Am

itri

ptyl

ine

Dilt

iaze

m

Chlo

rpro

maz

ine

Nor

trip

tylin

e (IS

)

LC/MS/MS Chromatograms of 100 ng/mL spiked whole blood sample after mini-QuEChERS extraction; AOAC (NaAc) and d-SPE (PSA)

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3x10

0.10.20.30.40.50.60.70.80.9

11.11.21.31.41.51.61.71.81.9

22.12.22.32.42.52.62.72.82.9

33.13.23.33.43.53.63.73.83.9

44.14.24.34.44.54.64.74.84.9

55.1

+ESI MRM Frag=112.0V [email protected] (278.2000 -> 117.0000) HQC_6.d

1 1

Counts vs. Acquisition Time (min)0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4 4.1 4.2 4.3 4.4 4.5 4.6

1x10

4.154.24.254.34.354.44.454.54.554.64.654.74.754.84.854.94.955

5.055.15.155.25.255.35.355.45.455.55.555.65.655.7

+ESI MRM Frag=112.0V [email protected] (287.1000 -> 240.9000) RE_L_1.d 1

Counts vs. Acquisition Time (min)3.053.13.153.23.253.33.353.43.453.53.553.63.653.73.753.83.853.93.9544.054.14.154.24.254.34.354.44.454.54.554.64.654.74.75

Signal-to-noise for Oxazepam and Lorazepam, at 10 ng/mL (10 ppb), SN = 3.0


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Example of mini-QuEChERS extracted standard linear curve for Naloxone from 10-250 ng/mL (ppb), R2 = 0.991.


Recovery and ReproducibilityCompound 25 ng/mL Spiked

Recovery RSD50 ng/mL Spiked

Recovery RSD100 ng/mL Spiked

Recovery RSD

Lidocaine 81.6 35.3 98.7 15.7 100 11.8

Tramadol 97.2 18.6 105 3.0 104 8.2

Amitriptyline 85 13.6 104 2.1 104 8.2

Biperidene 75.5 14.8 97 4.5 99 8.2

Oxazepam 60.4 17.3 77.0 9.2 78 8.6

Lorazepam 68.4 17.0 81.9 6.8 81.8 8.6

Chlorpromazine 75 14.1 110 10.3 105 6.3

Diltiazem 63.7 15.8 88.1 2.7 91.7 8.3

Naloxone 68 12.1 80.6 9.0 75.5 7.7

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Conclusion and Future Work• mini-QuEChERS approach for the extraction of pharmaceutical compounds

from whole blood offers an alternative to other sample preparation techniques

• mini-QuEChERS sample preparation is a simple, easy and cost effective approach, requiring minimal sample preparation expertise and equipment

• Extend the number and classifications evaluated

• Other biological matrices, postmortem blood• Future work will involve convenient form factors for both the mini-QuEChERS

extraction salts and d-SPE

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the quechers approach to determine pharmaceuticals and

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