Mechanism of renal excretion

Drugs not dependent on metabolism or biliary excretion for clearance will, in most cases, be renally cleared. Lipophilicity is an important parameter in governing the relative proportion of metabolic versus renal clearance. Increasing lipophilicity affects the binding of xenobiotics to the active site of many of the enzymes of drug metabolism, particularly the CYPs. As a result, increasing lipophilicity to log D74 > 0 will increase metabolic clearance, while at the same time, it will likely...

Standard addition method

If finding an effective sample preparation method with appropriate internal standards is too time-consuming, the standard addition method to correct for matrix effects can be tried. This method requires at least two LC-MS MS assays the first with the unknown sample (X) and the second with the unknown sample spiked with a known amount of standard (X + A). If the mass response is linear between unknown sample (RX) and spiked unknown sample (RX+A), the following relationship can be established as...

Retention time min

Figure 1.6 LC-MS MS chromatograms showing the use of a high-speed gradient the upper trace shows the standard assay for an analyte and its internal standard (IS) with a 4-min run time, while the lower trace shows the fast assay with a minibore column for the same two compounds with a 1-min run time. While the assay time was reduced from 4min to 1min, good chromatographic peak shapes for both the analyte and internal standard were maintained in the higher speed assay. Source Hsieh et al. 38 ....

Nature of matrices hydrophilic versus hydrophobic

Identifying the nature of matrices will provide useful information for overcoming the matrix effects. Most drug candidates are small molecules with log P ranging from 1 to 5 and are retained on a reversed-phase HPLC column.27 By manipulating pH, the composition of the mobile phases and the gradient46 or selecting a mini-bore column,47 one can easily separate the drug candidates from those polar and nonvolatile matrices that are typically eluted at an earlier retention time on a reversed phase...

Evaporation rate and ionevaporation model

Based on the ion evaporation model (IEM) of gas phase ion formation described by Irbarne & Thomson, Tang and Kebarle proposed an equation to predict ESI response of analyte A in the presence of electrolytes (E) and other components (M) using the evaporation rates and the concentrations.14,21,24 The typical equations are listed below. Three components IA fp, r ,., ka Aj I. (4.2) p A JFka A + km M + ke E v 7 As described by Tang and Kebarle, the product fp is a factor that was assumed to be...

Modifying the mobile phase

The ideal mobile phases for ESI are those with an optimum surface tension that facilitates the generation of a stable spray.67 For positive mode, the most popular mobile phases are methanol water or acetonitrile water or methanol acetonitrile water with a weak acid (such as acetic or formic) added to the solution at the concentration range from 0.005 to 0.05 , or with a weak acid buffer (such as formic acid-ammonium formate or acetic acid-ammonium acetate). The low pH of such mobile phases can...

References

Gilbert, J.D., Olah, T.V., Barrish, A., and Greber, T.F., Determination of L-654,066, a new 5 alpha-reductase inhibitor in plasma by liquid chromatography atmospheric pressure chemical ionization mass spectrometry, Bio. Mass Spectrom., 21, 341, 1992. 2. Ikonomou, M.G., Naghipur, A., Lown, J.W., and Kebarle, P., Characterization of the reaction products of deoxyguanosine with the anticancer agent BFNU and BFNU-1,1,1',1'-d4 in different buffers by high-performance liquid chromatography...

Acyl Glucuronide Formation

Acyl glucuronide metabolites are formed by the conjugation of the carboxylic acid moiety of a drug or metabolite with glucuronic acid. Glucuronidation is a major metabolic pathway for detoxifying and eliminating xenobiotics, including a wide range of hypolipidemic and NSAIDs in mammals. Acyl glucuronides contain an ester group that is susceptible to both hydrolysis and intramolecular acyl migration. Hydrolysis of an acyl glucuronide conjugate converts it to the aglycone which may be the parent...

The importance of integration of early DMPK data with other HTS data

Although multiple tools screens are available, the decision to employ a screen within a drug discovery project must come from a rational appraisal of the project requirements, rather than simply because that screen is capable of providing the needed throughput. Furthermore, the point must be made that any improvements in throughput are worthless unless they are supported by rigorous and continued validation of the overall screen performance 21 . The integration element of rational HTS is very...

Transfer supernatant to 96well plate using Tomtec Quadra

Figure 1.17 Semi-automated sample preparation procedure used in the CARRS assay. Adapted from Korfmacher et al. 87 . With permission. take 4-5 weeks can now be performed in a few days. Sample tracking can be performed using either an Excel (Microsoft Corp.) -based tracking system or a laboratory LIMS system such as WATSON (InnaPhase Corp., www. innaphase.com). Standard curve preparation can be readily performed using robotic sample handling systems (e.g., Packard MultiPROBE ) that can not only...

Hepatic uptake

Recent expression cloning approaches have revealed the presence of multiple members of the organic anion transporting polypeptide (OATP-1) family, Table 2.3 Transporter proteins involved in hepatic uptake of drugs Basic, zwitterionic and neutral compounds Basic, zwitterionic and neutral compounds Acidic compounds Basic, zwitterionic and neutral compounds Basic, zwitterionic and neutral compounds Small organic cations Bile acids, conjugated steroids (i.e. cdehydroepi-androsterone sulfate,...

Postcolumn infusion

In order to directly observe the location of ionization suppression in an LC-MS MS assay, Bonfiglio and colleagues51 developed a post-column infusion scheme that has been widely adopted by many laboratories. In this scheme, as presented in Figure 4.7, blank sample extracts are injected on the HPLC column under conditions chosen for the assay while a constant amount of analyte is infused into the HPLC stream before it enters the mass spectrometer. Ion suppression caused by matrices is shown as...

Prespiking and postspiking comparison

In order to separate the recovery loss from matrix suppression, one can use this pre-spiking and post-spiking approach, where pre-spiking refers to adding standards and internal standards before sample preparation and post-spiking refers to adding standards and internal standards after sample preparation.59,60 As demonstrated in Figure 4.9, response I was produced by the neat analyte solution, free of matrix effects and binding loss. Response II was obtained from pre-spiking procedure and...

Incomplete evaporation

It is a well-known fact that the presence of nonvolatile salts such as phosphate and sulfate in the mobile phase is deleterious for ion sources of LC-MS MS systems due to the deposition of solid material onto surfaces of the source. Nonvolatile components in biological sample can also cause significant ion suppression for early-eluting compounds. Ions that are generated in droplets can only be detected after they are emitted into the gas phase, therefore evaporation is a critical process for...

The evolving role of DMPK studies in drug design

By the end of the 1980s, it was becoming common practice to obtain PK metabolism data, if not in the design stage, then at least before the compound(s) advanced far into development. Initially, the PK metabolism data collected was predominantly whole animal data. As shown in Table 2.1 there is a natural inclination to this approach. In general, while whole animal studies are considered more physiologically relevant, they are also more expensive and time consuming than in vitro studies....

Experimental methods for determining Vd

Prediction of human PK remains a central goal of the drug discovery process. Given the importance of distribution to efficacy and duration of drug action, much effort has gone into predicting distribution. Among the various models used to predict drug distribution some have been purely theoretical, some have been experimental, or a hybrid of both. Most models center around three main approaches (1) QSAR-type models, (2) in vitro dialysis or (3) allometric scaling. As an example of the first...

Online Solidphase Extraction Procedures

Solid-phase extraction (SPE) has become one of the more popular techniques to remove interference materials in complex samples because of its simplicity, speed, and effectiveness. The SPE technique can be integrated into HPLC systems by column switching to provide for on-line sample extraction 13 . For on-line SPE, the biological samples are injected into the preconditioned cartridge or disposable pre-column (available under the brand names LiChrograph OSP-2 and PROSPEKT ) followed by distilled...

Competing for limited surface excess charge

Electrospray Ionization Images

Even though there are different mechanisms (IEM and CRM) for gas phase ion formation, both theories agree that initial gas ions are generated from the surface phase of ESI droplets. Both the ion-evaporation model (Equation 4.1) and the partitioning-equilibrium model (Equation 4.12) are used to predict that the ESI response in the presence of other components is based on competition for a fixed amount of ESI current (I) or excess charge (Q) which is controlled by the applied voltage and the flow...

Surface activity and partitioningequilibrium model

With the consideration of the importance of surface activity, Enke developed the partitioning-equilibrium model to predict the ESI response of an analyte with a single charge in the presence of matrix components based on the surface activity of the analyte and co-eluting components as well as the competition for the limited number of excess charge sites on the surface of the initial droplet without invoking the effect of ion evaporation.9 Excess charge on the surface of the Taylor cone and the...

Staggered semiparallel injection and analysis

Another way to reduce cycle time further is to implement the method of staggered segmented injections. In this method, multiple most often two autosamplers and columns are used in combination to eliminate common delays associated with sample loading and equilibration times. Staggered injection and elution techniques use two columns in parallel and acquire data in what is considered the useful part of chromatograms and can reduce overall analysis time as well. Wu applied this method to enhance...

Direct Plasma Injection using Restricted Access Media

Chart Medical Injections

Another promising packing material designed for direct extraction of small molecules in biological samples to allow multiple injections is the so-called restricted access media RAM . The working principle of RAM phases is to isolate macromolecules from the targeted small molecules in biological fluids based upon their particle sizes and chromatographic interaction. The large macromolecules such as proteins, which are unable to penetrate the hydrophobic pores and the hydrophilic outer layer of...

Indexed MUX parallel mass spectrometry

The commercialized multiplexed MUX electrospray interface, which introduces multiple LC flows directly into a multiplexed indexed electrospray ion source, has also been applied successfully for high throughput ADME applications as well 40, 41 . Yang et al. 41 identified the two main advantages of parallel LC-MS MS using a Micromass Ultima with MUX interface to be 1 parallel analysis and 2 four-times the throughput relative to single column systems. However, disadvantages were reported as 1...

Integration of DMPK data

Optimizing permeability, metabolic stability, solubility and other DMPK properties through structure modification while still maintaining potency can be quite daunting. This is primarily due to the opposing requirements of the properties for intestinal permeability, distribution, metabolism, and biliary or renal clearance. For example, increasing a compound's lipophilicity generally increases membrane permeability and receptor binding affinity, but at the same time, also may make the compound a...

Direct Monolithic Silica Chromatographic Systems

A useful approach to enhance column efficiency smaller H is to increase the column permeability, K r L AP, where r, L, and AP are linear velocity of mobile phase, solvent viscosity, column length, and pressure drop, respectively in a particle packed column. This can be achieved by using a monolithic silica column, a novel stationary phase with small-sized skeletons and large through-pores to simultaneously reduce the diffusion path length and flow resistance relative to a traditional, particle...

Mechanism of glucuronidation

Several mechanisms of acyl glucuronide reactivity have been proposed. The transacylation mechanism proposed by van Breemen and Fenselau results in covalent binding of the drug without the glucuronic acid and protein via nucleophillic displacement of the glucuronosyl group by NH2, SH or Rearrangements and reactions of acyl glucuronides Figure 6.1 Glucuronidation is one of the major phase II metabolic pathways of carboxylic acid compounds. Once formed, aycl glucuronides may undergo acyl migration...