Materials and Methods

2.2.1 Chemicals and Reagents

Lopinavir (LPV) and ritonavir (RTV) were provided by Abbott Laboratories (Abbott Park, Chicago, IL, USA). Darunavir (darunavir ethanolate, DRV) was provided by Tibotec (Mechelen, Belgium), and raltegravir (RAL) was obtained from Merck

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Sharp & Dohme (Haarlem, The Netherlands). Quinoxaline (6,7-dimethyl-2,3- di(2pyridyl)-quinoxaline, QX) was used as the internal standard (IS) and this was purchased from Sigma Aldrich (UK). LCMS grade acetonitrile (ACN) was purchased from Fisher Scientific (Loughborough, UK). HPLC grade methanol (MeOH) was purchased from VWR Laboratory supplies, UK. Formic acid and Hanks balanced salt solution (HBSS) were purchased from Sigma-Aldrich, UK. Deionised water was obtained from an Elga Option-S water purification unit (Elga LabWater, High Wycombe, UK) and then further purified to 18.2 MΩ with a Purelab Ultra unit (Elga LabWater, High Wycombe, UK). Ficoll was purchased from G.E Healthcare (Bucks, UK). Human peripheral blood mononuclear cells (PBMCs) were purchased from the National Blood Service (Liverpool, UK).

2.2.2 Equipment

The high performance liquid chromatography (HPLC) system consisted of a variable loop Accela autosampler (set to a temperature of 15oC) and an Accela LC pump (Thermo Fisher Scientific, Hemel Hempstead, UK). A reverse phase Ascentis™ C18

column (3μm: 100 mm×2.1 mm) set at an oven temperature of 30 o

C (Supelco, Dorset, UK), fitted with a 2μm C18 Quest column saver, (Thermo Fisher Scientific,

Hemel Hempstead, UK) was used to separate all analytes and internal standard. The HPLC system was interfaced with a Thermo Fisher Scientific TSQ Quantum Ultra triple stage quadrupole mass spectrometer, fitted with a heated-elecrospray ionisation (H-ESI) probe, housed in an ion max source. Two E2M30 rotary vacuum pumps (Edwards High Vacuum International, West Sussex, UK), a NM30LA nitrogen generator (Peak Scientific Instruments, Renfrew, UK), and research grade (99%

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pure) argon gas (10L size V, BOC, Manchester, UK) were used. Analyte tuning and optimisation parameters were gained with the use of TSQ Tune software (Thermo Fisher Scientific, Hemel Hempstead, UK). LC Quan TM software (Version 2.7, Thermo Fisher Scientific, Hemel Hempstead, UK) was used to acquire and process the data.

2.2.3 Chromatographic system

The injection mode was set at full loop (10l loop), therefore only requiring a sample volume of 10µL. The autosampler tray temperature was set at 15oC and the column oven control at which chromatographic separation took place was set at 30oC. Separation of the antiretrovirals and IS was facilitated by a gradient elution (Table 1). Mobile phase A consisted of 0.2% formic acid, and mobile phase B consisted of 100% ACN. The flow rate was constant at 400 L/min. At 6.1 minutes the mobile phase ratio was returned back to the starting conditions to allow equilibration of the column. The total run time for the assay was therefore 8 minutes. A wash solution containing ACN: water, 80:20, v/v, was used to clean the injection syringe (2 mL) between each sample injection. Both the mobile phase and the wash solution were de-gassed and mixed by sonication for 20 minutes prior to use. All analytes were scanned for using selective reaction monitoring (SRM) by the TSQ Quantum Ultra operated in positive ionisation mode.

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Table 1. Mobile phase gradient.

Time (min) Mobile Phase A % Mobile Phase B % Flow rate

(µL/min) 0 95 5 400 2 95 5 400 3.5 20 80 400 6 20 80 400 6.1 95 5 400 8 95 5 400

2.2.4 Mass spectrometric system

Each of the antiretroviral drugs and the IS were prepared in methanol at a concentration of 1µg/mL. This combined solution was then directly injected in to the mass spectrometer using a syringe pump (10 µL/min). The molecular mass (parent mass), for each of the analytes were scanned for in the first quadrupole (Q1). Once detected the tube lens was then optimised for each of the drugs (Fig 1). The parent ions then travelled to the second quadrupole (Q2) which contains inert gas molecules that fragment the ions into their product ions. Collision energy was applied at a range of voltages (v) (Fig. 2) and the product ions for each of the parent masses were scanned for in the selected reaction monitoring (SRM) mode in the third quadrupole (Q3). Two product ions for each of the parent ions were selected. These were the ions which gave the greatest mass to charge ratio (m/z) for their parent ion, with the least amount of background noise. The optimised mass spectrometric parameters are shown in table 2.

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Figure. 1. Optimisation of the tube lens in the first quadrupole. The previous setting show those optimised for an ARV plasma assay. Each ARV is shown in a different colour with its corresponding parent mass. RTV = 721.4 m/z, LPV = 629.3 m/z, DRV = 548.2 m/z, RAL = 445.1 m/z and QX (internal standard) = 313.1 m/z.

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Figure. 2. Optimisation of collision energy in the second quadrupole for each of the analyte parent ions, to fragment into the daughter ions. Each ARV daughter ion is shown in a different colour, outlined in the figure key. Table 2 outlines the ARV parent ions m/z and the fragment ions post collision energy.

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Table 2. Optimised conditions for the mass spectrometric detection of each of the analytes in positive ionisation mode.

Drug Name Retention Time (mins) Parent ion (m/z) Product ion (m/z) Spray voltage Vap temp Sheath gas pressure Aux gas pressure Capillary temp tube lens offset Coll pressure (mTorr) Collision energy (v) LPV 4.78 629.3 154.99 5000 350 60 40 300 77 1.5 37 429.13 5000 350 60 40 300 77 1.5 21 RTV 4.68 721.3 269.01 5000 350 60 40 300 95 1.5 27 296.01 5000 350 60 40 300 95 1.5 18 DRV 4.35 548.2 155.87 5000 350 60 40 300 95 1.5 33 392.73 5000 350 60 40 300 95 1.5 14 RAL 4.19 445.1 108.97 5000 350 60 40 300 83 1.5 33 361.01 5000 350 60 40 300 83 1.5 17 QX 4.3 313.1 246.04 5000 350 60 40 300 109 1.5 42 284.03 5000 350 60 40 300 109 1.5 45

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2.2.5 Source and chromatography maintenance

Concentrated constituents of the intracellular lysate may build up in the column guard, column and LC tubing. In order to circumvent this, the column and column guard were reverse flushed at a flow rate of 400 µL / minute, with 95% ACN and 5% water for 30 minutes after each analytical run. The Ultra source housing unit was removed and cleaned with 50% MeOH after each analytical run. The source cone and ion transfer tube were also removed and cleaned between each assay by immersion in 50% MeOH and sonicated for 20 minutes at room temperature.

2.2.6 Column re-equilibration

Prior to a new assay, the Ascentis™ C18 column was re-equilibrated. This was

achieved by manually controlling the LC pump conditions to the chromatography starting conditions for the assay (95% mobile phase A and 5% mobile phase B), with a flow rate of 400 µL / minute, for a period of at least 15 minutes. This was also sufficient to raise the source temperature to optimal conditions prior to the samples been run.