Direct analysis of quaternary amines and biphosphonate drugs in rat plasma by LC/MS/MS using a novel weak ion exchangers as on-line SPE sorbents

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Claude R. Mallet, Ziling Lu, Jeff. Mazzeo, Uwe Neue
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Other Symposium
Oasis MCX Extraction Column 2.1x20 mm Cartridge
Valethamate, Diphenzoquate, Propanolol, Quaternary Amines, Anticholinergic
Rat plasma, Human urine
XTerra MS C18 3.5 µm Steel 2.1 mm 30 mm
The use of strong ion exchangers can give a up to a 10 fold increase in sensitivity in on-line SPE LC/MS/MS. In a recent paper, we demonstrated 0.01 ng/mL LOQs using a novel combination of a mixed mode ion exchanger and reversed phase sorbent for on-line SPE LC/MS/MS [1,2]. The low LOQ are due to the use of a stronger clean up methodology in comparison to a less aggressive protocol with the stand alone reversed phase scheme. The fundamentals of on-line SPE using an ion-exchanger are quite simple. First, the basic or acidic drug is loaded as a neutral form on the extraction cartridge or column at high flow rate (4 mL/min). The drug will be trapped by the reversed phase mechanism of the mixed mode sorbent. Next, with a series of switching valves, several wash solutions can be utilize before the final elution step. The first wash step is crucial, for example, with a basic drug, an acidic aqueous solution is flushed through the cartridge to lock the drug on the ion exchanger. At this point, since the drug is safely trapped on the ion exchanger, stronger solvents can be used to clean the reversed phase of the mixed sorbent. The elution is done with a gradient with basic additives (i.e. ammonium hydroxide). This will neutralize the basic drug from the mixed mode sorbent Band the effluent can be directed toward an additional column for peak focusing or directly toward the mass spectrometer. However, if the targeted analytes are quaternary amines or biphosphonate drugs that can not be neutralized using the same strong ion exchanger would not yield any signal in both cases. The drugs would be strongly bound to the ion exchanger, meaning that a stronger eluent would be required for elution. For good retention of these type of molecules, most applications will use either an ion pairing agent [2] or ion exchange chromatography [4], which are not particularly suited for LC/MS/MS. Many have turned to CZE/MS to overcome these difficulties with the latter techniques. However, CZE/MS still lack sensitivity. A three valve configuration using one extraction column was used for the analysis of quaternary amine drugs in rat plasma and human urine. The on-line extraction was performed on a novel weak ion exchanger column (2.1 x 20 mm, 25 µm) using three Waters 515 pumps, a Waters 2700 auto sampler and a Waters 2690 in gradient mode. The extracted analytes were back flushed onto an XTerra® MS C18 column (2.1 x 30 mm, 3.5 µm, 186000398), added to provide additional separation power. The drugs were quantified using a Micromass Ultima™ triple quadrupole mass spectrometer equipped with an electrospray source and set in multiple reaction monitoring mode.