Analytical Techniques for Low-Dose Compounds

Again the point is that, for any given molecule, the choice of analytical technique may vary. The techniques outlined below may, in general, provide the highest probably of technical success for very stringent acceptance limits. In addition, the techniques are listed in the order of preference for cleaning verification assays that would have an acceptance limit of 50ng/swab (most potent compounds in Table 15.2) or lower, and this will be the focus of the remainder of this chapter. 

For pharmaceutical compounds, LC-MS has found extremely wide acceptance due to the low-level detection that can be achieved, in addition to the selectivity and specificity that are attained by using HPLC in conjunction with MS detection. LC-MS is also convenient because of its compatibility with reversed-phase HPLC mobile phases. Volatile mobile phase additives such as trifluoroacetic acid, formic acid, and ammonium hydroxide are very common and can be utilized not only to aid in the chromatographic separation but also to influence the ionization state of the molecule (i.e., acid modifiers to protonate [M + H]+1, and basic modifiers to deprotonate [M — H] ). This requirement may require a modification of the potency method if phosphate was utilized however, it is not viewed as a major drawback. 

In a work by Liu et al.,26 a cleaning-verification assay was validated for a highly potent family of compounds utilizing a swab-sampling procedure and LC-MS for separation and detection of the analytes. Due to the high potency of the compound, the LC-MS method was validated at a level of 50ng/25cm2 and 50 ng/100 cm2 

TABLE 15.5 Chromatographic Conditions Used to Obtain the Chromatogram in Fig. 15.3 

Mobile phase 80% methanol/20% pH 4.5 ammonium acetate buffer 

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To date, there is limited published material concerning the pharmacokinetics of vanadium compounds in humans. The concentration of vanadium in humans not dosed with the metal is extremely low and at the limits of detection of many of the analytical techniques used. It is not possible to ascertain if the large differences observed in different populations are the result of environmental exposure or experimental variability. Studies using blood have shown vanadium levels of 0.4 to 2.8 pg/L in normal people. The serum contains the largest amount of vanadium with concentration values ranging from 2 to 4 pg/L using atomic absorption spectroscopy [90], The upper limit of vanadium in the urine of normal people was reported to be 22 pg/L, with excretion values averaging below 8 pg/24 h. Vanadium is widely available in nutrition stores for athletes, who believe it to be a nonsteroidal compound that increases muscle mass at a dose of approximately 7 to 10 mg day, without any reports of toxicity [91]. 

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