15 Chromatography
Instrument Methods Basic Analytes LC Conditions: Table 1. LC system conditions for chromatographic separation of basic analytes.
LC system
Agilent LC-MS, consisting of a 1260 LC and Single Quadrupole Mass Spectrometer.
Computer running chromatographic software (OpenLabs)
Column
Column temp. Injection volume
Flow rate
Mobile phase A Mobile phase B
Raptor Biphenyl 30 x 2.1mm, 1.8µm 45°C
2.00 µL 600 µL/min
0.1% Formic acid in water 0.1% Formic acid in methanol Time (min) A%
Solvent Composition
0.00 4.30 7.00 7.01 10.0
95.0 57.5 57.5 95.0 95.0
Table 5. Properties for the compounds analysed - a Value from Drugbank b Predicted value from Pubchem *Compound has multiple ionisable groups present.
Compound Caffeine Procainamide
Compound Class
Formula
Neutral C8H10N4O2 Base
Acetaminophen Neutral Hydrocortisone
Prednisolone Pindolol Neutral Base Neutral
C8H9NO2 C21H30O5
C21H28O5 C14H20N2O2 Dexamethasone Neutral C22H29FO5
B% 5.0
42.5 42.5 5.0 5.0
Acidic Analytes LC Conditions: Table 2. LC system conditions for chromatographic separation of acidic analytes.
LC system
Agilent LC-MS, consisting of a 1260 LC and Single Quadrupole Mass Spectrometer.
Computer running chromatographic software (OpenLabs)
Column
Column temp. Injection volume Flow rate
Mobile phase A Mobile phase B
Solvent Composition
Raptor Biphenyl 30 x 2.1mm, 1.8µm 30°C
2.00 µL 400 µL/min
0.1% Formic acid in water 0.1% Formic acid in methanol
Time (min) A% B% 0.00
45.0
Neutral Analytes LC Conditions: Table 3. LC system conditions for chromatographic separation of neutral analytes.
LC system
Agilent LC-MS, consisting of a 1260 LC and Single Quadrupole Mass Spectrometer.
Computer running chromatographic software (OpenLabs)
Column
Column temp. Injection volume Flow rate
Mobile phase A Mobile phase B
Raptor Biphenyl 30 x 2.1mm, 1.8µm 30°C
2.00 µL 400 µL/min
0.1% Formic acid in water 0.1% Formic acid in methanol
Solvent Composition
Time (min) A% 0.00 5.10 6.00 6.01
12.00
Mass spectrometry parameters for all methods: Table 4. Mass spectrometer conditions for all classes of analytes.
Parameter
Gas Temperature Gas Flow Nebuliser
Capillary Voltage
Fragmentor Voltage Scan Type Ion Mode
Value 350 ºC
13 L/min 30 psi
4000 V 100 V SIM ESI
B%
70.0 25.0 25.0 70.0 70.0
30.0 75.0 75.0 30.0 30.0
Sample Preparation Methods Plasma preparation
Three separate quantities of pig plasma (Sigma-Aldrich) were spiked to a concentration of 1 µg/mL with basic, acidic and neutral compounds (Table 5) and allowed to equilibrate for 30 minutes before being used in both the SLE and LLE procedures.
SLE
Different pre-treatment solutions were used to dilute the plasma after each plasma sample was loaded into the well. This changes the pH of the solution to allow for optimal extraction with organic solvent on elution whilst also diluting samples to allow better fl ow onto the plate. For the pre-treatment solutions, basic compounds were diluted in 5% ammonia in water, acidic compounds in 2% formic acid in water and neutral compounds used deionised water. Neutral compounds are unaffected by pH so to ensure optimum recoveries, water was used to dilute the sample for better fl ow onto the Microlute™ SLE plate. 200 µL of the diluted plasma (100 µL of pig plasma + 100 µL of pre-treatment solution) was loaded onto the Microlute™ SLE 200 mg plate (Cat no: PSLE200P-001) using 3 PSI of positive pressure for 5 seconds (Figure 3). It was then allowed to load fully for 5 minutes to allow the sample to completely absorb.
To extract the analytes from the SLE product, 2 x 500 µL of solvent (see Table 1 for solvent used for each type of analyte) was added to each well and allowed to elute under gravity into a 1 mL collection plate (Cat no: 219250). Once both elutions were completed, 10 PSI of positive pressure was applied to the plate for 30 seconds to
complete elution. The eluent was evaporated to dryness using N2 with the Porvair Sciences Ultravap® Levante (Cat no: 500226) at 35°C and reconstituted in 200 µL of starting mobile phase as seen in the solvent composition sections of Tables 1, 2 and 3.
55.0 Ibuprofen Acid C13H18O2 206.3 3.97a 5.30 Nifl umic acid Acid C13H9F3N2O2 282.2 4.43a Corticosterone Neutral
4-Propylbenzoic acid
Naproxen Acid C21H30O4 C10H12O2
Molecular Mass
Log P 194.2 -0.07a C13H21N3O 235.3 0.88a
151.2 0.91a 362.5 1.61a
360.4 1.62a
248.3 1.75a 392.5 1.83a
346.5 2.02b 164.2 2.34b pKab
14.00 9.32
-4.40
12.59, -2.80*
12.59, -2.90*
9.25
12.42, -3.30*
13.86, -0.26*
4.40 Acid C14H14O3 230.3 3.04b Extraction Solvent Ethyl acetate
3:1 Hexane:Ethyl acetate
Ethyl acetate Ethyl acetate
Ethyl acetate
Ethyl acetate Ethyl acetate
Ethyl acetate 95:5
Dichloromethane/ IPA
4.10
4-Pentylbenzoic acid
Ketoprofen Acid C12H16O2 192.3 3.12b 95:5
Dichloromethane/ IPA
4.40 Acid C16H14O3 254.3 3.12a 95:5
Dichloromethane/ IPA
4.45 Propranolol Nortriptyline Base Base C16H21NO2 C19H21N 259.3 3.48a 263.4 3.90a 95:5
Dichloromethane/ IPA
9.42 9.70
3:1 Hexane:Ethyl acetate
3:1 Hexane:Ethyl acetate
95:5
Dichloromethane/ IPA
1.90, 5.50*
Diclofenac Acid C14H11Cl2NO2 296.1 4.51a 4.15 Protriptyline Imipramine Desipramine Amitriptyline Base Base Base Base C19H21N C19H24N2 C18H22N2 C20H23N 263.4 4.70a 280.4 4.80a 266.4 4.90a 277.4 4.92a 95:5
Dichloromethane/ IPA
95:5
Dichloromethane/ IPA
10.50 9.40 10.40 9.40
3:1 Hexane:Ethyl acetate
3:1 Hexane:Ethyl acetate
3:1 Hexane:Ethyl acetate
3:1 Hexane:Ethyl acetate
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