Tài liệu HPLC for Pharmaceutical Scientists 2007 (Part 13) docx

In recentyears the sensitivity and selectivity of MS/MS analysis of xenobiotics havebeen put to use in toxicokinetics, pharmacokinetics, metabolic, formulation,and early drug discovery s

ver-in complex mixtures Herever-in, we present an overview of the above techniquesaccompanied with several examples of the use of liquid chromatography–tandem mass spectrometry in pharmacokinetics/drug metabolism assessmentduring drug development.

Since the evolution of pharmaceutical research [1, 2], the stages of drug covery and development have followed three predominant patterns: (i) thesystematic and methodical approach by chemists to rationally design and syn-thesize a molecule to target a specific molecular system (e.g., ion channels,receptors, enzymes, DNA); (ii) the isolation and purification of the activeingredients of medicinal plants or microorganisms to screen their spectrum of

dis-605

HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons, Inc.

activity using in vitro models; or (iii) the serendipitous discovery of a

com-pound with a novel pharmacological action (e.g., the accidental discovery ofantidepressants) Today, one of the increasingly popular and complementaryapproaches for drug discovery in the pharmaceutical industry is to performmassive parallel synthesis in solution or on a solid support In addition, withthe advent of functional genomics and proteomics, cell-based assays, and mol-ecular biology, a multitude of therapeutic targets have been validated [3].With an increasing number of potential molecular targets identified throughthe science of functional proteomics and genomics, diverse libraries of newchemical entities (NCEs) have to be generated and evaluated Consequently,the rapid growth of combinatorial libraries has posed a need for faster,accurate, and sensitive analytical techniques capable of large-scale high-

throughput screening (HTS) Although in vitro assays do not necessarily reflect the complexity of the in vivo interactions, the speed and simplicity of

the former have rendered them an integral part of the screening process

In recent years, the in silico and experimental modeling of pharmacokinetic/

pharmacodynamic (PK/PD) relationship have become increasingly popular [4, 5] The integration of PK (i.e., drug dose and biological fluid concentration)and PD (i.e., pharmacologic effect) provides a key determinant in under-standing the dosing regimen and therapeutic effect of a potential drug com-pound To this end, analytical assays also play a pivotal role in defining thePK/PD relation of NCEs In many cases, both the drug concentration and PD

biomarkers (vide infra) can be directly measured in peripheral fluids using

specific analytical techniques

Furthermore, samples generated from large-scale clinical trials along withthe ambitious development timelines to get safe and efficacious drugs tomarket warrant the use of HT bioanalysis Numerous improvements in speed,sensitivity, and accuracy, augmented with innovations in automation in con-junction with mass spectrometry (MS) detection, have allowed for versatileand multifaceted platforms [6–8]

13.2 IONIZATION PROCESSES

Mass spectrometry (MS) is playing an increasingly visible role in the lar characterization of combinatorial libraries, natural products, drug meta-bolism and pharmacokinetics, toxicology and forensic investigations, andproteomics Toward this end, electrospray ionization (ESI), atmospheric pres-sure chemical ionization (APCI), and atmospheric pressure photo-ionization(APPI) have proven valuable for both qualitative and quantitative screening

molecu-of small molecules (e.g., pharmaceutical products) [9–14]

The utility of ESI (Figure 13-1) lies in its ability to generate ions directlyfrom the solution phase into the gas phase The ions are produced by appli-cation of a strong electric field to a very fine spray of the solution containingthe analyte The electric field creates highly charged droplets whose subse-

606 THE ROLE OF LC–MS IN PK AND DRUG METABOLISM

quent vaporization (or desolvation) results in the production of gaseous ions.The fact that ions are formed from solution has established the technique

as a convenient mass detector for liquid chromatography (LC/MS) and for automated sample analysis In addition, ESI-MS offers many tangible benefitsover other mass spectrometric methods including the ability to qualitativelyanalyze low-molecular-weight compounds, inherent soft-ionization, excellentquantitation and reproducibility, high sensitivity, and its amenability toautomation

Analogous to the ESI interface, APCI (Figure 13-2), also referred to as the heated nebulizer (HN), induces little or no fragmentation to the analyte

IONIZATION PROCESSES 607

Figure 13-1 A simplified schematic of the ESI process (Courtesy of Dr P Tiller.)

Figure 13-2 A simplified schematic of the APCI process (Courtesy of Dr P Tiller.)

Therefore, the APCI spectrum also tends to be simpler in interpretation thanthe traditional electron ionization (EI), which results in extensive fragmenta-tion of the precursor ion As a result, APCI and ESI are referred to as “soft-ionizations,” while EI is considered a “hard-ionization” technique Generally,volatile and thermally stable compounds can be subjected to LC/APCI/MSanalysis In quantitative analysis, APCI provides a greater (i.e., in terms of lin-earity) dynamic range than ESI and it is considered rugged, easy to operate,and relatively tolerant of higher buffer concentrations (i.e., fewer matrixeffects) In ESI, at about 10–5M and higher, the ion signal becomes fixed andindependent of sample concentration (plateauing effect) and may exhibit non-linearity at higher concentrations In contrast, APCI can offer a wider lineardynamic range For example, in our laboratory (data not shown) we have rou-tinely developed reversed-phase LC/APCI/MS/MS assays ranging from 1.0 ng/mL to 10,000 ng/mL with a correlation coefficient of >0.996 Further-more, APCI can accommodate flow rates of up to 2.0 mL/min and is effective

in the analysis of medium- and low-polarity compounds [12] In qualitativedrug metabolism studies, a combination of APCI and ESI experiments canprove valuable in distinguishing certain oxidative biotransformations (e.g.,

N -oxidation versus hydroxylation) [15, 16] In contrast to ESI, APCI is not

suited for the analysis of biopolymers, proteins, peptides, and thermally labilespecies

In the APCI process, electrons originating from a corona discharge needleionize the analyte via a series of gas-phase ion-molecule reactions Forexample, in the positive-ion mode, the energetic electrons start a sequence ofreactions with the nebulizing gas (typically nitrogen), giving rise to nitrogenmolecular ions Using APCI, depending on the composition of the HPLCmobile phase, ions such as [H2O + H]+, [CH3OH+ H]+, [NH3 + H]+, and/or[CH3CN+ H]+are formed via series of ion-molecule reactions with the nitro-gen molecular ions Subsequently, additional ionization is initiated by exother-mic proton transfers from the protonated solvent ions to the neutral analytemolecules yielding [analyte + H]+, [analyte + CH3OH+ H]+, [analyte + NH3+H]+ions, and so on In general, metal adduct ions are observed less commonly

in APCI as opposed to ESI, where they are more prevalent Greater ity is attained if the solvent is polar and contains ions through the addition of

sensitiv-an electrolyte The desolvation process is then further enhsensitiv-anced by the heatingelement within the APCI assembly, which is maintained at 300–550°C One ofthe drawbacks of APCI is its lack of compatibility with low eluent flow rates.The stability of the ionization response may be poor at low rates (i.e., less than50µL/min)

In contrast, ESI is compatible with miniaturized columns and amenable tosample-limited scenarios such as biochemical and biotechnological applica-tions ESI can be considered a flow-sensitive technique The dimension of theprimary droplets is dependent on the flow rate Therefore, by using columnswith a smaller internal diameter (i.d.) and consequently lower flow rates,the concentration of the analytes in the spray solution can vary and it can be

608 THE ROLE OF LC–MS IN PK AND DRUG METABOLISM

considered a dependent ionization process It is dependent in the sense that the surface charge density of the droplets in thegas phase is higher due to more effective desolvation of the droplets sincelower flow rates are used The use of solvent-buffer post-column addition alsoallows optimization for improved analyte ion current response Increasing theflow rate increases droplet size, which decreases the yield of gas-phase ionsfrom the charged droplets.

concentration-Recently, atmospheric pressure photo-ionization (APPI) [17–19] was duced as a complementary ionization technique to ESI and APCI APPI(Figure 13-3) is now commercially available by several MS vendors such asAgilent Technologies, Applied Biosystems (Sciex), Waters (Micromass), andThermo Electron (Finnigan) Corporations This technique can be used toionize an analyte that otherwise is not easily ionizable using either APCI orESI In APPI, to increase ionization efficiency, a high-intensity UV radiationsource is used (i.e., a 10-eV krypton discharge lamp) in a direct or an indirectmode In the direct mode, often a molecular ion is generated by irradiation;while in the indirect mode, a dopant is used in conjunction with the analysis

intro-A photoionizable dopant such as acetone or toluene is employed to mediate(as dopant photo-ions) the production of ions by proton or electron transfer.The dopant is introduced to the APPI ionization chamber by a separate pump

at an optimized steady flow rate during analysis (e.g., 10–15% of the mobilephase flow rate, post-column) A number of excellent articles have recentlybeen published on the applicability of APPI for the analysis of small mole-cules [17–19]

Source Block Dopant

13.3 TANDEM-MASS SPECTROMETRY (MS/MS)

For purposes of quantitative analysis, selected ion monitoring (SIM) andselected reaction monitoring (SRM) are two commonly utilized approaches.The latter is also referred to as multiple reaction monitoring (MRM) In bothmodes, considerable structural information is lost; nonetheless, these tech-niques are extremely powerful for target compound quantification in biolog-ical matrices, if the compound of interest is known

In the SIM mode, the MS is tuned to a particular m/z window (preferably

at unit resolution), which corresponds to the ion of interest (i.e., [M + H]+, or

a stable adduct such as [M + X]+, where X = Na, K, NH4, etc.) SIM may require

a more elaborate chromatographic separation in order to minimize ence from endogenous species However, in the SRM approach, higher selec-tivity and sensitivity are realized Thus, shorter chromatographic runs (fasterinjection cycles) and limited sample pretreatment could be tolerated withoutsignificant loss in sensitivity

interfer-In addition, due to lack of MS/MS capability, SIM has been more commonlyperformed on single quadrupole MS, while SRM has been broadly adapted ontriple quadrupole (Figure 13-4) and ion-trap mass spectrometers The increase

in sensitivity and selectivity of SRM stem from the ion-chromatogram (i.e.,LC-MS/MS) obtained by specific precursor-to-product ion transition for ananalyte of interest (Figure 13-4) Conversely, in an SIM mode, the relativebackground noise due to the presence of other isobaric species (i.e., ions with

a same m/z as the analyte of interest) can result in a lower signal-to-noise ratio

for the analyte Due to the widespread acceptance of SRM in quantitativeanalysis, the remaining part of this section focuses on a description of tandem-mass spectrometry (MS/MS), which is utilized in SRM (or MRM) experiments.Tandem-mass spectrometry or collision-induced dissociation (CID) is one

of the most widely used techniques for probing the structure of ions in the gasphase [20] To this end, ease of application to various instrumental types, alongwith its experimental simplicity, account for the wide popularity of CID In a

typical CID experiment, a beam of ions with a specific m/z (denoted as the

precursor or parent ion) is selected and collided with a neutral and tive gas-phase target (e.g., argon, xenon, helium, nitrogen) These collisionsresult in subsequent fragmentation and product ions that are a direct conse-quence of dissociation of the precursor ion Generally, the resulting fragmen-tation pattern is unique to a particular ion structure The various CIDtechniques can be subdivided into categories based on the translational or collision energy of the precursor ion prior to collision with the target gas Thetwo main categories include low-energy CID, in the range of 1–300 eV (i.e.,used in triple quadrupole and ion-trap instruments), and high-energy CID atapproximately 1–25 KeV (i.e., used in guided-ion beam or sector instruments).Currently, one of the most common approaches is to perform MS/MS exper-iments on a triple quadrupole instrument Tandem-MS experiments have beenparticularly popular for the qualitative and quantitative analysis of small mol-

nonreac-610 THE ROLE OF LC–MS IN PK AND DRUG METABOLISM

ecules such as pharmaceutical products in biological fluids [21–23] In recentyears the sensitivity and selectivity of MS/MS analysis of xenobiotics havebeen put to use in toxicokinetics, pharmacokinetics, metabolic, formulation,and early drug discovery studies.

13.4 SAMPLE PREPARATION USING AN OFF-LINE APPROACH

One of the critical steps in qualitative and quantitative analysis is the samplepreparation procedure Sample preparation step can affect specificity, sensi-tivity, accuracy, precision, and throughput of a bioanalytical procedure In addi-tion to development and optimization of the chemistry involved in sampleprocessing, the use of semiautomated or fully automated protocols has been

SAMPLE PREPARATION USING AN OFF-LINE APPROACH 611

Figure 13-4 Representative MRM scans (plasma extract of a proprietary compound)

using an API 5000 triple quadrupole unit (Sciex) Each panel contains a distinct MRMtransition for the same compound: m/z 1021.6 → 1003.5 (left panel) and m/z 1021.6 →971.5 (right panel) Signal-to-noise ratio is designated as S/N Experimental conditions:ESI, positive ion mode, protein precipitation was used for sample preparation, injec-tion volume was 10µL, the column was a C18, and dimension was 20 × 2.1 mm, using alinear gradient elution: 0 min (20% B)–6 min (90% B)–8 min (90% B), where B was0.2% formic acid in acetonitrile and A was 0.2% formic acid in water; separation wasperformed at room temperature

implemented in recent years [24, 25] The popularity of off-line sample cessing in batch-mode has dramatically improved the throughput of this rate-limiting step.

pro-Generally, there are three commonly used approaches for off-line sampleprocessing: SPE (solid-phase extraction), LLE (liquid–liquid extraction), andprotein precipitation (PPT) These three methods have been successfully used

in conjunction with robotics for achieving an increase in sample preparationthroughput For example, Figure 13-5 is the photograph of a Beckman’sBiomek 2000 (other models such as Biomek 3000 and Biomek FX are alsoapplicable) for semi-automated sample preparation that can accommodateSPE, LLE, and PPT procedures This scheme has been established for use withSPE, LLE, or PPT in a 96-well plate format to analyze pharmaceutical prod-ucts in biological matrices (e.g., whole blood, plasma, serum, and cerebralspinal fluid (CSF)) in our laboratories (unpublished data)

13.4.1 SPE

In the 96-well SPE format, similar to the traditional manual procedure, issuessuch as the nature of the bonded-phase (e.g., ion exchange, C2, C8, C18, cyano,phenyl, polymeric, strong or weak cation exchange, strong or weak anionexchange, mixed phases, etc.), solvent strength (for conditioning/washing ofthe phases, target analyte elution), and chemical characteristics (e.g., solubil-ity, presence of the key functional groups) of the analyte(s) need to beaddressed A general scheme for initial development of an SPE method is out-

612 THE ROLE OF LC–MS IN PK AND DRUG METABOLISM

Figure 13-5 Photograph of a Biomek 2000 setup for semiautomated PPT, LLE,

or SPE process in the authors’ laboratory (also see www.beckmancoulter.com and reference 99)

lined below Depending on the structure of the compound (hydrophobicityand ionizable functionalities), specific steps to optimize sample recovery areneeded.

• Condition sample for optimum retention

• Condition SPE bed with methanol

• Equilibrate SPE bed with water

• Load sample onto SPE bed

(a) Cation exchange: wash with 2% formic acid [low pH (3)]

(b) Anion exchange: wash with 50 mM NaOAc buffer [high pH (8–10)]

• Wash bed with 5% methanol

• Elute retained materials with an organic solvent (i.e., CH3OH, CH3CN,isopropyl alcohol, or a combination thereof)

(a) Cation exchange: add 5% NH4OH to eluent

(b) Anion exchange: add 2% formic acid to eluent

Some of the most commonly utilized robotic modules for the 96-well SPEprocedure are Tomtec Quadra (Tomtec, Hamden, CT, USA), Packard Multi-Probe (Packard Instruments, Meriden, CT, USA), Biomek (Beckman–Coulter,Fullerton, CA), and Tecan (Durham, NC, USA) units

For example, we have successfully and routinely adopted the TomtecQuadra technology in the development and validation of several off-line SPEassays in whole blood, plasma, and urine followed by MS detection ThePackard Multi-Probe liquid handling workstation (Figure 13-6) has also shownpromise for off-line SPE procedures involving plasma and serum [26–28]

In addition, this unit as well as the Tecan and Biomek systems can be grammed for the initial sample (e.g., plasma) transfer step from vials to the96-well blocks, buffer addition (if applicable), and to aliquot internal standard.The advantage of the above capabilities is a significant reduction in time andlabor for the entire sample processing procedure Possible technical problemssuch as carry-over by fixed-tip pipettes used to aliquot the biological fluid can

pro-be alleviated by incorporation of several wash cycles or their replacement withdisposable pipette tips In addition, possible inaccurate transfer of samplesfrom the collection tubes to the 96-well blocks due to pipette tip clogging byendogenous protein clots or lipid layers should also be considered Specificsteps such as storage of the plasma samples at −80°C and/or centrifugation at14,000 rpm prior to sample transfer can be considered for precluding fibrino-gen clot formation

13.4.2 PPT

Due to its ease of use and speed, PPT is one of the most common approaches

in sample preparation in early drug discovery [25] While PPT is fast, apply, and applicable to a broad class of small molecules, it also suffers from

easy-to-SAMPLE PREPARATION USING AN OFF-LINE APPROACH 613

several disadvantages Briefly, in a PPT procedure, often an equal or highervolume (e.g., 1 : 3) of acetonitrile (or sometimes methanol) is added to a sample

of plasma, which contains the test article as well as an internal standard Thesample is mixed and centrifuged, resulting in the formation of a protein pelletand its corresponding supernatant The supernatant is transferred, dried,reconstituted, or directly injected onto a LC column Clearly, this procedure

is easily amenable to automation and is applicable to a host of structurally

614 THE ROLE OF LC–MS IN PK AND DRUG METABOLISM

Figure 13-6 Photograph (top panel) of a Packard Multi-Probe (www.perkinelmer.

com) platform for semiautomated PPT, LLE, or SPE process in the authors’ tory The bottom panel shows a typical layout using the corresponding operating software package

labora-diverse group of small molecules However, PPT lacks specificity and tivity that SPE or LLE can offer Consequently, significant matrix effect andion suppression can be observed due to the presence of other endogenous mol-ecules that compete with the analyte(s) during ionization [29–38] In addition,compounds that are highly bound to the protein can yield low sample recov-ery in the PPT procedure PPT procedure is also more demanding on the MSinterface, which requires more frequent cleanup, due to the endogenous inter-ference and contaminants An example of PPT in drug development will be

selec-presented in the latter part of this chapter (STI571; vide infra).

13.4.3 LLE

Liquid–liquid extraction is another well-established and attractive approach,which has been useful for the analysis of xenobiotics in biological fluids LLEcan be designed to be highly selective yielding cleaner sample extracts Briefly,LLE is a mass transfer procedure where an aqueous sample (e.g., analyte con-taining biological fluid) is in contact with an immiscible solvent that exhibitspreferential selectivity toward one or more of the components in the aqueous

sample (e.g., plasma or whole blood) In an SPE procedure (vide supra), a solid

sorbent material such as an alkyl-bonded silica is packed into a cartridge, into

a disk, or in a 96-well plate format, and it performs essentially the same tion as the organic solvent in LLE This is particularly critical in minimizingion suppression by co-eluting matrix components, when an ESI interface isused for the LC/MS analysis Due to a different mechanism of operation, ionsuppression is not a major determinant for signal loss in APCI [37–40]) Theion suppression is exacerbated in some cases, where the chromatographyresults in low peak capacity factors [39] This could be attributed to co-elutionwith polar species that had also partitioned into the immiscible solvent andwere consequently injected onto the HPLC column Based on a series ofexperiments reported by King and co-workers [35], the order of ESI responsesuppression is PPT > SPE > LLE, where liquid–liquid extraction yields theleast amount of analyte ion loss

func-13.5 AUTOMATED SAMPLE TRANSFER

Lastly, one of the labor-intensive steps in bioanalytical sample processing isthe accurate initial transfer of plasma or whole blood from cryogenic vials(e.g., polypropylene tubes) to 96-well plates This step is particularly laboriousand time-consuming when a large number of samples (e.g., in 1000s) are sub-jected to analysis in late-stage clinical trials The main bottleneck involves the

“manual” uncapping and re-capping steps for each individual vial In thisregard, the Tomtec Corporation is in the process of final testing and commer-cialization of the “Formatter” (Figure 13-7), which is designed to alleviate theabove bottleneck According to the vendor (and tests during a demo by one

AUTOMATED SAMPLE TRANSFER 615

of the authors), this unit will be able to accurately de-cap, aliquot, transfer,and re-cap automatically In addition, the unit can pipette 10–450µL accu-rately, has sample bar code tracking capability, and can interface with Watson-LIMS (www.tomtec.com) In a typical experiment, plasma samples aretransferred from individual vials to a 96-well plate and subsequently processed

by an appropriate extraction method on a Tomtec unit (PPT, SPE, LLE) Ofcourse, steps such as vortexing and centrifugation still require a manual inter-vention; hence, sample extraction methods using a Tomtec are often referred

to as “semiautomated.”

13.6 SAMPLE PROCESSING USING AN ON-LINE APPROACH

In recent years, high-throughput and automated on-line sample extractionprocedures have offered viable alternatives to improve efficiency for sampleprocessing [41–46] One such approach has been turbulent flow LC In turbu-lent flow LC, single- or dual-column configurations have commonly beenreported Recently, a four-channel staggered injection system (e.g., Cohesive’smultiplexing Aria LX-4 system) has been reported for decreasing the MS idle time and improving productivity (cycle time) [45] A typical dimension

of the extraction column can be 50 × 1.0 mm (i.d.), although smaller lengthscan also be used In the single-column configuration, a sample containing the analyte and internal standard is loaded on the extraction column at a

616 THE ROLE OF LC–MS IN PK AND DRUG METABOLISM

Figure 13-7 Photograph of a Tomtec “Formatter” (a prototype) designed to de-cap,

aliquot, and re-cap sample vials containing biological fluids (e.g., plasma, serum, wholeblood) For more details see www.tomtec.com

high linear velocity (e.g., 5.0 mL/min) The analyte is retained via rapid sion into the packing, while other matrix components are washed into wasteusing an aqueous mobile phase Subsequently, the analyte is eluted by a step or linear gradient and is detected by the mass spectrometer In the dual-column configuration, a standard analytical column (e.g., C18or C8; 50- ×4.6-mm i.d.) is placed after the extraction column to improve chromatographicseparation and sample cleanup In our laboratory, we have successfully validated and applied the dual-column (Figures 13-8 and 13-9) configuration

diffu-to perform racemic reversed-phase as well as chiral LC-MS/MS analysis

In the latter assay, we replaced the second column by one containing a chiralstationary phase A full account of the assay optimization and validation

of an on-line achiral–chiral column configuration has been reported where [47]

else-Direct sample injection has also been accomplished by using on-line C18

(4-mm i.d.) guard cartridges for cytochrome inhibition studies, capillary SPE, C18alkyl-diol-silica restricted access phase, and PROSPEKTTM SPE modules Amore recent instrument by Spark–Holland (Figure 13-10, the Symbiosis unit)consists of Shimadzu HPLC pumps, “conditioned stacker,” an autosamplerhousing, and a “high-pressure dispenser” SPE station [48, 49] This unit can beused as a stand-alone autosampler (LC mode) and/or an on-line sample-processing module (XLC mode) with a mass spectrometer The companionsoftware is embedded in the Sciex’s Analyst® 1.4.1 and can be easily usedwithin the sample batch design and as part of its acquisition method The

-vendor also provides 96-well plate-screening cartridges containing HySphere

SPE resins such as C18, C8, C2, CN, ion exchange, and so on, in a 12 × 8 format(dimension: 2 × 10 mm)

SAMPLE PROCESSING USING AN ON-LINE APPROACH 617

Figure 13-8 Simplified schematic of two-valve/two-column system (built in-house)

used in the turbulent flow LC experiment [47] The first column (e.g., Cyclone P, 50 ×0.5 mm) is used for the extraction step In our laboratory, this design has been used inconjunction with chiral, standard narrow bore (e.g., 50 × 2 mm), and monolithic ana-lytical columns

618 THE ROLE OF LC–MS IN PK AND DRUG METABOLISM

Figure 13-9 Configuration of a dual channel on-line sample extraction, the CTC Trio

Valve system (LEAP Technologies, www.leaptec.com; also see reference 100 for nical details), which is commercially available AC and PC signify analytical and pro-cessing columns, respectively

tech-Figure 13-10 Photograph of a Spark–Holland system, which can be used in the off-line

(LC) and on-line SPE (XLC) modes This unit contains an autosampler, two Shimadzupumps, a degasser, and SPE capability (for more details see www.sparkholland.com)

A caveat for all direct sample injection assays is an understanding of theanalyte chemical stability in the biological fluid during the analysis period.Nonetheless, an increasingly growing body of literature is suggestive thatdirect injection of post-dose biological fluids for quantification purposes hasbecome a routine and efficient procedure.

13.7 MATRIX EFFECT AND ION SUPPRESSION

Ion suppression or so-called matrix effect is a common problem in pheric pressure ionization (API) mass spectrometry [29–40] There are differ-ences in opinion as to the amount of ion suppression that is acceptable for ananalytical method

atmos-In some laboratories, ion suppression in an analytical method is not able, but in other laboratories it is acceptable if there is no significant effect

accept-on the validity of the analytical data with appropriate quality caccept-ontrols Thephenomenon of ion suppression results in reduction of signal intensity Con-sequently, the lower limit of quantification (LLOQ) for highly sensitive bio-analytical methods could be difficult to achieve The problem can be furthercomplicated by (a) differential ion suppression due to intersubject variabilityand (b) the use of blank bio-matrix with varying lot numbers (i.e., controlblood obtained from different patients/subjects) in preparing the calibrators.Differences in ion suppression between the analytes and structurally differentinternal standards may also be problematic This issue can be mitigated by theuse of stable-isotope-labeled (SIL) analogues as internal standards The extent

of ion suppression is dependent on the methods for sample preparation andchromatographic separation The supernatant produced by the PPT method ismost likely to cause an ion suppression in ESI due to the lack of selectivity ofPPT procedure (co-eluting endogenous compounds such as lipids, phospho-lipids, fatty acids, etc., that affect the ESI droplet desolvation process; for moredetails see reference 38 and technical notes on www.tandemlabs.com) Theextracts obtained from solid-phase extraction (SPE) and liquid–liquid extrac-tion (LLE) are relatively cleaner Further studies on the molecular identities

of co-eluting endogenous compounds, leading to ion suppression, are required

to clearly delineate their contribution

One of the widely used methods to qualitatively assess the matrix effectconsists of post-column addition of analytes to the LC-eluent flowing from thecolumn to the ESI interface of the mass spectrometer (Figure 13-11) [35].Briefly, an analyte and the internal standard (IS) dissolved in the same LCeluent are infused (e.g., flow rate 10µL/min) using a syringe pump, through a

“tee-mixer,” located between the column eluent (e.g., flow rate 200µL/min)and the ESI interface of the mass spectrometer An extract (using LLE orSPE) or supernatant (if PPT is used) from an analyte-free matrix, such asblank or control plasma, is injected via the autosampler, while the test articleand the internal standard are introduced, post-column, to the MS ionization

MATRIX EFFECT AND ION SUPPRESSION 619

source at a stable and continuous flow using an infusion pump Since the testarticle and the internal standard are introduced to the MS at a constant flow,

a steady ion response is obtained as a function of time If there is an ion pression, a drop in the MS ion signal is observed upon the injection of theextract obtained from a control plasma (or any other biological matrix) Theinfusion LC-MS/MS (MRM mode) extracted ion chromatograms of both the internal standard and the analyte are shown in Figure 13-12 for an off-linePPT plasma supernatant injection In this example there is no significant ionsuppression from 3–10 min If there is a significant ion suppression, modifica-tion to a more selective (cleaner) sample preparation, adoption of a more elab-orate chromatographic condition to separate ion suppressing agents from theanalyte(s) of interest, and/or use of a stable label internal standard are rec-ommended [29–40] Figure 13-13 illustrates the significance of the choice ofsample preparation on signal-to-noise ratio of an investigational compound,where an OASIS®-HLB sorbent (hydrophilic–lipophilic balanced co-polymer)(denoted as HLB in Figure 13-13) SPE and a strong anion-exchange SPE(MAX) (denoted as anion ex in Figure 13-13) yielded higher ion intensity thanPPT.The count per second (CPS) signifies the detector (an electron multiplier)response More efficient extraction was obtained with the anion-exchange SPEthan with C18SPE or using the PPT mode

sup-13.8 REGULATORY REQUIREMENTS FOR LC/MS

METHOD VALIDATION

Validation of quantitative LC/MS methods used in the determination of molecule drugs and/or their metabolites in biological fluids is of paramount

small-620 THE ROLE OF LC–MS IN PK AND DRUG METABOLISM

Figure 13-11 The post-column infusion experiment commonly used for the qualitative

assessment of ion suppression and originally reported by King and co-workers [35] (MPsignifies mobile phase)

importance and a key determinant in obtaining reliable pharmacokinetics(PK) information A properly developed and validated LC/MS method mayoften be used throughout the process of a drug’s evaluation lifecycle Thesecould include early discovery PK studies, preclinical toxicology studies (e.g.,dose-proportionality studies), salt/formulation selection (pharmaceuticalresearch and development), clinical PK studies, and post-marketing surveil-lance (Figure 13-14) [50–53] Hence, intra- and interlaboratory specificity,accuracy, precision, and ruggedness have to be established [54].

In order to bridge some of the regulatory filings (e.g., within the UnitedStates, Japan, and Europe), the US Food and Drug Administration (FDA)Center for Drug Evaluation and Research (CDER), the International Conference on Harmonization (ICH), Japan’s regulatory agency, and theEuropean Community have devised common as well as distinct requirementsfor bioanalytical method validation [55] To this end, it is imperative to strictlyfollow these requirements during preclinical toxicology (e.g., toxicokinetics)and Phases I, II (a and b), and III clinical trials as well as during all the post-marketing PK studies (Figure 13-15)

Moreover, in introduction of generic or new formulations and/or to lish bioequivalence (BE) between two products, certain guidelines are fol-lowed to compare the systematic exposure of the test article to that of a

estab-REGULATORY REQUIREMENTS FOR LC/MS METHOD VALIDATION 621

Figure 13-12 Representative MRM scans obtained using the ion-suppression infusion

experiment, developed by King and co-workers [35] A significant ion matrix effect isobserved between 0.5 and 1 min using control rat plasma The sample preparation wasPPT Note that this experiment needs to be performed “prior” to the method devel-opment and validation, so necessary changes to the sample preparation protocol andchromatographic method are made Reprinted with permission from [101]

622 THE ROLE OF LC–MS IN PK AND DRUG METABOLISM

Figure 13-13 Comparison of choice of sample preparation on MRM signal intensity

of an investigation compound The injection volume was 40µL The count per second(CPS) signifies the detector (an electron multiplier) response Protein precipitation(PPT), hydrophilic–lipophilic balanced co-polymer-based SPE (Oasis HLB- co-

polymer of styrene, divinylbenzene and n-vinylpirrolidone monomers; the hydrophilic

refers to the NVP monomer, and the lipophilic refers to the SDVB monomers), andstrong anion exchange SPE (Max) (all in 96-well plate format) were used in controlrat plasma (unpublished data)

Figure 13-14 A generic flow chart for the process of drug discovery and

develop-ment [102]