Handbook of LC-MS Bioanalysis (Best Practices, Experimental Protocols, and Regulations) || LC-MS Method Development Strategies for Enhancing Mass Spectrometric Detection

31LC-MS METHOD DEVELOPMENT STRATEGIES FORENHANCING MASS SPECTROMETRIC DETECTION

Yuan-Qing Xia and Jeffrey D. Miller

31.1 INTRODUCTION

Robustness of liquid chromatography–tandem mass spec-trometry (LC-MS/MS) bioanalytical methods is critical inproviding support for accurate assessment of toxicokineticsand pharmacokinetics of drug candidates in various stagesof discovery and development (Jemal and Xia, 2006; Jemalet al., 2010). Many aspects are involved in developing andvalidating a robust LC-MS/MS bioanalytical method andmost of those aspects have been captured in respective chap-ters of this book. In the present chapter, we would like todiscuss signal enhancement in mass spectrometric detectionin LC-MS/MS bioanalysis. In particular, we would like todiscuss differential mobility spectrometry (DMS) and mul-tiple reaction monitoring cubed (MRM3) for reducing highchemical background noise, eliminating matrix interferencesand separating isomeric and metabolites in quantitative LC-MS/MS bioanalysis. Furthermore, we would like to illus-trate strategies for enhancing the mass spectrometric signalintensity and selectivity by utilizing either atmospheric pres-sure photoionization (APPI) or mobile phase additives andemploying anionic and cationic adducts as analytical precur-sor ions in the LC-MS/MS bioanalysis process.

31.2 DIFFERENTIAL MOBILITYSPECTROMETRY

DMS, also referred to as high field-asymmetric waveformion mobility spectrometry (FAIMS), is a variant ion mobil-ity spectrometry (IMS). Conventional IMS separates ionsaccording to their mobility through gas phase under the

constant electrostatic field of low field strength (Purvesand Guevremont, 1999; Guevremont, 2004; Kolakowski andMester, 2007; Schneider et al., 2010). For DMS, ions arepulsed into the flight tube and their flight times are recorded.The drift time, the ion mobility, is a function of the reducedmass, charge state, and shape of an ion through its inter-actions with the background gas. DMS differs from IMSin the geometry of the instrumentation and separates ionsvia employing the field dependence of the coefficient of ionmobility. In brief, the separation of ions in DMS is based onanalyte-specific differences in ion mobility under the influ-ence of a high electric field (RF voltage), known as the sep-aration voltage (SV), and a low electric field of a waveformapplied to the two electrodes. Due to the difference betweenhigh and low field ion mobility coefficients, ions will migratetoward the walls and leave the flight path unless their trajec-tory is corrected by a counterbalancing DC voltage, knownas the compensation voltage (COV). The COV thus controlswhich ions are transmitted through DMS, and gas is usedto propel the ions through the electrodes. Thus, the drift ofan ion toward either electrode is based on the difference inthe mobility of that ion in high and low fields and is analytespecific parameter. The term DMS is used to distinguish thistechnique from IMS. Thus, the resolution of ions by DMS isbased on the difference between the COV values of the ions.DMS, when used in conjunction with LC-MS/MS, acts asa postcolumn and premass spectrometer ion filter in whichonly selected ions generated from electrospray ionization(ESI) or atmospheric pressure chemical ionization (APCI)sources are transmitted. Ideally, when the selected COV isoptimum for the analyte, the ions of the analyte pass throughthe DMS cell into the orifice of a mass spectrometer, while

Handbook of LC-MS Bioanalysis: Best Practices, Experimental Protocols, and Regulations, First Edition. Edited by Wenkui Li, Jie Zhang, and Francis L.S. Tse.C© 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc.

391

392 LC-MS METHOD DEVELOPMENT STRATEGIES FOR ENHANCING MASS SPECTROMETRIC DETECTION

(a)

(b)

RF

(vo

lts)

1400

1200

1000

800

600

Ion speciesin gas stream

– Ions

+ IonsCV

Comp.voltage

Positive (+)

RF = 1000 v

–30 –20 –10 0vc

–30 –20 –10 100vc

Negative (–)

SV

Or to MS

FIGURE 31.1 Schematic of DMS ion filter and sensor opera-tion. (a) Ions generated in an atmospheric pressure ion source arecarried in a transport gas through a DMS analysis region of theapplied fields (SV and CV), and then detected at Faraday platedetectors. (b) Dispersion plots, recorded for dimethyl methylphos-phonate (DMMP). In this experiment, SV (vertical) was scannedbetween 500 and 1500 V and CV (horizontal) between −40 and+ 10 V. Positive ions (left) and negative ions (right) were recordedsimultaneously. Positive ions are separated into three ion species:reactant ions peak (RIP), DMMP monomer, and DMMP dimer.Right frame shows negative background ion behavior (reproducedfrom Schneider et al. (2010) with permission of Elsevier).

the ions of the background chemical ions or interferences arefiltered away before reaching the orifice. Consequently, DMSprovides separation between the analytes and the backgroundinterferences even in the absence of chromatographic sepa-ration. Only the ions with specific mobility dependence willpass through the electrodes of the device. This process typi-cally occurs in the order of tens of milliseconds. The abovecombined feature allows bioanalytical scientists to signifi-cantly reduce interferences or background matrix ions dueto the presence of isobaric compounds, coeluting species, orendogenous components in LC-MS/MS bioanalysis.

As shown in Figure 31.1, the DMS mobility cell iscomposed of two flat plates that are parallel to one anotherand define the mobility region. The ions are drawn bythe transport gas flow toward the mass spectrometer.SelexIONTM technology (AB Sciex) couples the DMS

device to the QTRAP R© 5500 or QTRAP R© 6500 massspectrometer, in which the two DMS electrodes are sealed tothe inlet orifice with a juncture chamber in between. A gasport in the chamber allows full control of the residence timeand resolution within the DMS device. The ion mobilitycoefficient is encoded in the compensation voltage and isused to correct the tilt in ion trajectory of each SV amplitude.The compensation voltage is scanned serially to pass ionsaccording to their differential mobility, or set to a fixed valueto pass only the ion species with a particular differentialmobility. SelexIONTM technology has short residence times,which enable rapid voltage changes for MRM operation.Due to the short MRM cycle times (20 ms pause time), DMScan couple with ultrahigh pressure liquid chromatographyfor fast LC analysis. The DMS device shows minimaldiffusion losses and improved resolution at high voltages.In addition, it can simply be used in transparent mode(MRM mode) allowing all ions to be transmitted by turningoff SV and COV voltages without the need to physicallyremove the DMS device. Figure 31.2 shows the comparisonof LC-MS/MS chromatograms with and without DMS forthe analysis of testosterone in plasma samples preparedvia protein precipitation using acetonitrile. With DMS off,both MRM transitions of m/z 289 to m/z 109 and m/z 289to m/z 97 show significant endogenous matrix interferencepeaks coeluting with testosterone. With DMS on, theendogenous matrix interference peaks were eliminated andthe chromatograms of the same two MRM transitions showan excellent analyte peak with very clean baseline. As aresult, the peak integration for testosterone was much easierfor quantitation. Figure 31.3 illustrates an excellent exampleusing DMS to enhance the full scan MS selectivity forsafranin by eliminating chemical background of polyethy-lene glycol. With DMS off, when acquiring full scan Q1MSvia infusing safranin containing PEG 400, the target mass ofsafranin (m/z 315) was not detectable due to the high back-ground PEG 400 ions. However, with DMS on, an enhancedMS spectrum of safranin (m/z 315) was clearly detectedwhile the PEG 400 ions were filtered out before entering themass spectrometer. By using DMS, safranin was selectedpassing through the DMS cell, but not the PEG 400 ions, dueto the different characteristic ion mobility of the analytes.

In LC-MS/MS bioanalysis, one of the potential pitfalls isthe MS in-source conversion of metabolites to parent drugdue to in-source fragmentation or conversion. In-sourceconversion of metabolites, especially phase II metabolites(glucuronide and sulfate), can generate the same drugmolecular ions, which can hamper the accurate quantitativebioanalysis of the drug if there is no LC separation betweenthe drug and its phase II conjugates (Jemal and Xia, 1999).Xia and Jemal (2009) applied FAIMS to separate ifetrobanfrom its acyl glucuronide metabolite. The optimized COVvalue was determined as −13.7 V for ifetroban and −10.7 Vfor its acyl glucuronide, respectively. When injecting the acyl

MULTIPLE REACTION MONITORING CUBED 393

DMS offm/z 289/109

DMS onm/z 289/109

3.37

S/N = 136Peak Int.(Subt.)=1.4e+4

3xStd.Dev.(Noise)=1.1e+2

S/N = 155.9Peak Int.(Subt.)=1.8e+4

3xStd.Dev.(Noise)=1.2e+2

–Noise –Noise

3.37

DMS offm/z 289/97

DMS onm/z 289/97

Testosterone Testosterone

2.0e4 2.5e5

2.0e5

1.5e5

1.0e5

5.0e4

0.0

1.4e41.8e4

1.6e4

1.4e4

1.2e4

1.0e4

1.2e4

1.0e4

1.8e4

1.6e4

1.4e4

1.2e4

1.0e4

8000.0

6000.0

4000.0

2000.0

0.0

8000.0

6000.0

4000.0

2000.0

0.0

8000.0

6000.0

4000.0

2000.0

0.0

2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0Time (min)

2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0Time (min)

2.4 2.5

3.13

3.16

3.30

3.41

3.49 3.86

2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0Time (min)

2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0Time (min)

2.82

3.03

3.16

3.50

3.85

3.31

3.37

Inte

nsity

(cp

s)

Inte

nsity

(cp

s)

Inte

nsity

(cp

s)

Inte

nsity

(cp

s)

(a)

(b) (d)

(c)XIC of + MRM (3 pairs): 289.200/109.200/Da ID: Testoster... XIC of + MRM (3 pairs): 289.200/97.100/Da ID: Testosteron...

XIC of + MRM (3 pairs): 289.200/97.100/Da ID: Testosteron...XIC of + MRM (3 pairs): 289.200/109.200/Da ID: Testoster...

Max. 2.0e4 cps.

Max. 1.5e4 cps. Max. 1.8e4 cps.

Max. 2.5e5 cps.

FIGURE 31.2 LC-MS/MS chromatograms of testosterone in human plasma sample extract pre-pared via protein precipitation using acetonitrile: (a) Deferential mobility spectrometry (DMS) offwith MRM of m/z 289→m/z 109, (b) DMS on with MRM of m/z 289→m/z 109, (c) DMS off withMRM of m/z 289→m/z 97, and (d) DMS on with MRM of m/z 289→m/z 97.

glucuronide metabolite with the optimized ifetroban COVvalue of −13.7 V, a small ifetroban (Figure 31.4a) peakand acyl glucuronide peak were observed (Figure 31.4b).This demonstrated that the acyl glucuronide metabolite ionswere filtered away at COV of −13.7 V. Thus, the in-sourceconversion of the acyl glucuronide metabolite to its parentdrug occurred almost entirely after the orifice of the massspectrometer, with the little conversion observed in thesource chamber.

31.3 MULTIPLE REACTION MONITORINGCUBED

A typical MRM process is to select a precursor ion in Q1MS,and fragment the precursor ion in the collision cell in thepresence of collision gas with a suitable collision energyapplied. Then a product or daughter ion is selected in Q3MSfor detection. Sometimes, matrix and/or endogenous inter-ference may share the same MRM transition as the analyteof interest. If the interference peaks coelute with the analyte,the selectivity of LC-MS/MS is hampered. This can lead to

overestimation of the analyte of interest in the biologicalsamples from the intended study. Several approaches haveto be taken to address the issues. These approaches include(1) revamping sample preparation method to eliminate theinterference in the sample extract prior to LC-MS/MS injec-tion, (2) chromatographic separation of the analyte of interestfrom the interference, and/or (3) employing additional frag-mentation in MS/MS detection with a linear ion trap (Figure31.5). Apparently, the last one is the easiest if assay sensi-tivity is reachable. This additional fragmentation is referredto a MRM3 scan, which utilizes the selection of a precur-sor ion in Q1MS, fragmentation of the precursor ion inthe collision cell, and selection of a fragment ion in Q3(linear ion trap, LIT) to further fragmentation to generatesecondary fragment ions. An MRM3 ion chromatogram isreconstructed from selected specific fragment ions producedfrom a primary product ion trapped in the Q3 linear iontrap and subsequently activated by resonant excitation. Sim-ilar to DMS, linear-ion-trap instrument with MRM3 can bevery useful for LC-MS quantitation of analytes in complexbiological matrixes with greatly reduced background noiseand/or endogenous interferences (Fortine et al., 2009). In


Max. 9.9e6 cps.+Q1: 0.657 min from sample 12 (safranin orange in peg) of jan 12 2011.wiff(turbo spray)

Safranin orange

(a)

100%

80%

60%

40%

20%

0%100

121.2 133.1 177.2216.9239.4

155.2195.0 209.3

225.1 276.2 329.2 338.5376.2 391.4

415.4437.4 481.3 525.3 569.5 613.2 637.2 681.4 725.5 765.2 786.5

315.2

261.3 283.4

305.2 327.2

341.3 359.2

349.3

371.3

409.2416.4

415.3

393.2437.3

459.4

481.2

525.3

503.3

547.3

569.4

613.4

635.4

591.3

573.4

657.4

679.3701.5

739.5

541.4497.3476.4453.3

150 200 250 300 350 400m/z (Da)

450 500 550 600 650 700 750 800

100 150 200 250 300 350 400m/z (Da)

450 500 550 600 650 700 750 800

100%

80%

60%

40%

20%

0%

Rel

. Int

. (%

)R

el. I

nt. (

%)

(b)+Q1: 0.085 to 0.304 min from sample 12 (safranin orange in peg) of jan 12 2011.wiff(turbo spray)

DMS off

DMS on

Max. 6.4e5 cps.

FIGURE 31.3 Positive full scan mass spectra of safranin containing polyethylene 400 (PEG 400)acquired using QTRAP R© 5500 with scan range of m/z 100–800 with (a) DMS off and (b) DMS on.

1.0 1.5 2.0 2.5 3.0 3.5Time (min)

0

50,000

100,000

150,000

200,000

250,000

300,0000

50,000

100,000

150,000

200,000

250,000

300,000

Inte

nsity

AA: 77796AA: 75875

AA: 44087

m/z 441 > m/z 423

m/z 617 > m/z 423

Leaked glucuronide metabolite under CV = 13.7 V)

BMS-180291Impurity from Ife-Glu)

In-source CID generated

(a)

(b)

ifetroban from glucuronide metabolite of ifetroban

FIGURE 31.4 LC-FAIMS-SRM chromatograms obtained from the injection of a sample contain-ing only ifetroban acylglucuronide (Ife-Glu) with the CV set at −13.7 V (optimum CV value forifetroban): (a) ifetroban SRM channel (m/z 441 → m/z 423); (b) Ife-Glu SRM channel (m/z 617 →m/z 423) (reproduced from Xia and Jemal (2009) with permission of John Wiley & Sons, Ltd).

MULTIPLE REACTION MONITORING CUBED 395

Q1MS Q2: Collision cell Q3-Linear ion trap

FIGURE 31.5 Schematic of multiple reaction monitoring cubed (MRM3) scan: selection of aprecursor ion in Q1MS, fragmentation of precursor ion in the collision cell, and selection of afragment ion in Q3 (linear ion trap) for further fragmentation to generate secondary fragment ions.

practice, suitable speed of MRM3 scan is necessary to allowfor the second generation product ions to be extracted andintegrated for quantitative bioanalysis. LIT instruments, suchas QTRAP R© 5500, 6500, and 4500, are commercially avail-able to meet these needs. Xu et al. (2009) compared MRMversus MRM3 for the quantitation of exenatide in plasma.With MRM transition of m/z 838 to m/z 396, several sig-nificant peaks of plasma matrix interferences were coelutedwith exenatide (Figure 31.6a), demanding an elevation ofthe lower limit of quantitation (LLOQ) from 5 ng/ml to

25 ng/ml for the robustness of the assay. With MRM3 (LC-MS/MS/MS) of m/z 838→m/z 396→m/z 202, the coelutinginterference peaks were eliminated and an excellent signal-to-noise ratio was seen for the analyte at the LLOQ of5 ng/ml (Figure 31.6b). On the other hand, due to the inter-ference of the coeluting components, a nonlinear calibrationregression has to be used for exenatide with MRM method(Figure 31.6c). With MRM3 method, a linear calibration stan-dard curve was achieved for the analyte with no difficulty(Figure 31.6d).

(a)

(b)

MRM838/396

MS3

838/396/202

MRM3

5 ng/ml

5 ng/ml

(c) Standard curve using MRM

(d) Standard curve using MRM3

FIGURE 31.6 LC-MRM chromatograms of exenatide in plasma sample extract: (a) MRM of m/z838 to m/z 396, (b) MRM3 of m/z 838→m/z 396→m/z 202, (c) with a quadratic calibration curvefor the standards using MRM of m/z 838 to m/z 396, and (d) with a linear calibration curve for thestandards using MRM3 of m/z 838→m/z 396→m/z 202.


31.4 ATMOSPHERIC PRESSUREPHOTOIONIZATION

In LC-MS/MS bioanalysis, the most widely used ionizationtechniques are ESI and APCI, which have been used in anal-ysis of polar and easily ionizable compounds but may notapplicable for nonpolar or less ionizable molecules. As acomplement to ESI and APCI techniques, APPI can be usedto expand the range and classes of compounds that can be ana-lyzed by LC-MS toward less polar compounds (Kostiainenand Kauppila, 2009). The principal benefit of APPI, as com-pared to other ionization sources, is the efficient ionizationof classes of nonpolar compounds, such as polychlorinatedbiphenyls (PCBs) and compounds with naphthalene moi-eties, which are not easily ionizable under conventional ESIor APCI. In APPI, a solvent is vaporized with a heated nebu-lizer, but the ionization process is initiated by using a vacuumultraviolet lamp instead of a corona discharge needle as seenin APCI. APPI is less susceptible to ion suppression and saltbuffer effects than APCI and ESI. The ionization process ofAPPI can be direct ionization or solvent-mediated reaction.The former takes place without addition of a solvent, while adopant (such as toluene) is needed for the latter. The dopantserves as an extra solvent for initiating and enhancing the ion-ization. In direct APPI, the initial reaction is the formation ofa molecular ion (M + •) by photoionization of the analyte,which must possess ionization energy below the energy of thephotons. In the presence of a protic solvent (methanol, water,2-propanol, cyclohexane), the molecular ion of the analyteabstracts a hydrogen atom from the solvent to form a proto-nated molecule (Hanold et al., 2004; Kauppila et al., 2002).In the dopant-assisted mode, a large excess of a dopant, suchas toluene or acetone, is added. The dopant molecule firstundergoes photoionization and then acts as a charge car-rier for subsequent ionization of trace levels of the analyte.Hanold et al. (2004) conducted comparison of ESI-CE-MSand APPI-CE-MS in analysis of a drug mixture consisting ofterbutaline, salbutamol, and labetalol. They concluded thatAPPI gives significantly better detectability (e.g., signal-to-noise ratio) than ESI primarily due to reduced noise by theAPPI source.

31.5 MS SIGNAL ENHANCEMENT VIA MOBILEPHASE ADDITIVES AND ANIONIC AND CATIONICADDUCTS PRECURSOR IONS

The effect of mobile phase composition on the ionization effi-ciency of an analyte of interest in ESI and APCI may not beeasily predicted. Therefore, achieving optimal LC-MS/MSconditions may take a great effort due to the complexity ofionization processes and many factors that affect the LC-MS/MS bioanalytical assay. In LC-MS, characteristics ofsolvents and solvent additives, including volatility, surface

tension, viscosity, conductivity, ionic strength, pH and gasphase ion–molecule reactions, may all contribute to ioniza-tion and signal responses of analyte of interest. MS detectionsensitivity is also influenced by chemical and physical prop-erties of the analyte, including pKa, hydrophobicity, surfaceactivity, and proton affinity. The effect of eluent composi-tion on the ionization efficiency of ESI, APCI, and APPIin LC-MS has been reviewed by Kostiainen and Kauppila(2009). In general, nonvolatile buffers such as phosphateand borate tend to cause increased background, signal sup-pression, and contamination of the ion source, resulting indecreased sensitivity and reproducibility. The most widelyused aqueous mobile phases contain acetic acid, formic acid,trifluoroacetic acid (TFA), ammonium bicarbonate, ammo-nium hydroxide, ammonium acetate, and/or ammonium for-mate. In practice, the concentration of these additives shouldnot exceed 20 mM in order to avoid suppression of ionizationand reduction of sensitivity. Typical MS “friendly” organicmobile phases include acetonitrile and methanol. In mostcases, mobile phase additives and buffered mobile phase areneeded for the resolution, peak shape, and/or retention ofanalyte of interest in liquid chromatography. On the otherhand, the chemical properties and concentration of the mobilephase additives have a significant effect on analyte responsein ESI, APCI, and APPI.

Mallet et al. (2004) studied the influence of several addi-tives and their concentrations on the ESI responses of acidicand basic drugs. The results showed a clear decrease inthe response when the concentration of the additive suchas formic acid, acetic acid, TFA, ammonium formate, andammonium bicarbonate was increased from 0.05% to 1%.They also reported that ammonium formate and ammoniumbicarbonate have a stronger suppression effect than that ofacidic (formic and acetic acid) and basic buffers (ammo-nium hydroxide) on the ESI response of selected modeldrugs. Kamel et al. (1999) reported that the addition of 1%acetic acid resulted in a good LC separation and enhancedMS detection sensitivity in positive ion mode for a seriesof nucleosides, while the addition of 50 mM ammoniumhydroxide resulted in a decreased sensitivity in negative ionmode. Duderstadta and Fischer (2008) reported that the sig-nal intensity for polyalkene additive compounds in APCIhas been found to be highly dependent upon the type of theorganic solvent used in the LC separations. When employinga water/methanol gradient in place of a water/acetonitrile ora water/acetone gradient, the analyte signal intensities wereincreased between 2.3-fold and 52-fold.

TFA has frequently been used in the LC analyses of basiccompounds. TFA not only controls the pH of the mobilephases but also acts as an ion-pair agent to improve peakshapes of basic compounds on silica-based columns. TFAis volatile and, therefore, can be used in LC-MS of proteinsand peptides as well as small molecules (Garcı́a, 2005; Shouand Naidong, 2005). The major drawback of using TFA in

MS SIGNAL ENHANCEMENT VIA MOBILE PHASE ADDITIVES AND ANIONIC AND CATIONIC ADDUCTS PRECURSOR IONS 397

0

20

40

60

80

0.1 uM 1 uM 10 uM 100 uM

Formic acid concentration Acetic acid concentration

A: HCOOH

compound Icompound IIcompound IIIcompound IV

1 mM 10 mM 100 mM 1M

100

% in

tens

ity

120

140

160

0

20

40

60

80

0.1 uM 1 uM 10 uM 100 uM

Propionic acid concentration n-Butyric acid cocentration


1 mM 10 mM 100 mM 1M

100

% in

tens

ity

120

140

160

0

20

40

60

80

0.1 uM 1 uM 10 uM 100 uM

B: CH3COOH

C: CH3CH2COOH


1 mM 10 mM 100 mM 1M

100

% in

tens

ity

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140

160

0

20

40

60

80

0.1 uM 1 uM 10 uM 100 uM


1 mM 10 mM 100 mM 1M

100%

inte

nsity

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140

160

D: CH3CH2CH2COOH

FIGURE 31.7 Effects of carboxylic acids on the negative-ion ESI responses of four SARMs. Thehorizontal axis represents the final concentration of modifier in the flow before entering the ESIsource. The vertical axis represents the mean (SD, n = 3) ratio of peak area of each compound inthe presence of modifier to the peak area of each compound in the absence of modifier multiplied by100% (reproduced from Wu et al. (2004) with permission of John Wiley & Sons, Ltd).

LC-MS, however, is that TFA suppresses the ESI signals ofanalytes and reduces assay sensitivity. This is primarily dueto the ability of TFA to form gas-phase ion pairs with posi-tively charged analyte ions (Shou and Naidong, 2005). Thesuppression of ionization by TFA resulting in unstable sprayhas been demonstrated in several studies. The spray instabil-ity and signal reduction were due to the high conductivityand surface tension of the aqueous eluent including TFA(Chowdhury and Chair, 1991) or strong ion pairing betweenthe TFA anion and the protonated molecule. The ion-pairingprocess is described as masking the protonated moleculesand thereby decreasing the efficiency of the ESI droplet toemit protonated molecules to the gas phase (Kuhlmann et al.,1995). Ion pairing may also lead to reduced charge separa-tion at the tip of the ESI sprayer and thereby to decreaseionization efficiency (Storm et al., 1999).

The most common method to overcome this issue involvesthe postcolumn addition of a mixture of propionic acid andisopropanol (Kuhlmann et al., 1995). However, the postcol-umn addition setup requires additional pumps and is notdesirable for continuous analysis of large amounts of sam-ples. Shou and Naidong (2005) reported a simple yet veryeffective means of minimizing the negative effect of TFAin LC-MS/MS bioanalysis by direct addition of 0.5% aceticacid or 1% propionic acid to mobile phases containing either0.025% or 0.05% TFA. A factor of two- to fivefold signalenhancement was achieved for eight basic model compounds.Furthermore, chromatography integrity was maintained evenwith the addition of acetic acid and propionic acid to existingTFA mobile phases.

Wu et al. (2004) studied the effects of various mobilephase modifiers on the negative ESI response of four


0

20

40

60

80

0.1uM 1uM 10 uM 100 uM

Ammonium formate concentration


1 mM 10 mM 100 mM 1M

100

% in

tens

ity120

A: HCOONH4

0

20

40

60

80

0.1uM 1uM 10 uM 100 uM

Ammonium acetate concentration


1 mM 10 mM 100 mM 1M

100

% in

tens

ity

120

A: CH3COONH4

FIGURE 31.8 Effect of neutral salts on the negative-ion ESI responses of four SARMs. Thehorizontal axis represents the final concentration of modifier in the flow before entering the ESIsource. The vertical axis represents the mean (SD, n = 3) ratio of peak area of each compound inthe presence of modifier to the peak area of each compound in the absence of modifier multiplied by100% (reproduced from Wu et al. (2004) with permission of John Wiley & Sons, Ltd).

selective androgen receptor modulators that do not havecarboxylic acid or any other strongly acidic group. Theyfound that acetic, propionic, and butyric acid at lowconcentrations from 0.1 μM to 10 mM improved theresponses of the analytes to varying degrees (Figure 31.7).In contrast, formic acid decreased the MS response of thetest compounds (Figure 31.7), as did ammonium formate,ammonium acetate, ammonium hydroxide, and triethylamine(Figure 31.8).

Molecules with strong acidic/basic functional groups, per-manent charge moieties and/or suitable surface activity usu-ally exhibit intense ESI signals. In contrast, molecules with-out presence of these structural attributes tend to respondpoorly in ESI detection and are therefore difficult to be ana-lyzed by LC-ESI-MS/MS, especially in complex biologi-cal matrices. One method to improve the signal responseof those analytes is the use of solvent adduct ions as pre-cursors for MS/MS detection. For example, anionic adductions can be employed as a precursor ion in negative ESImode to increase the LC-MS/MS sensitivity. Cai and Cole(2002) conducted a systematic investigation on the attach-ment of small anions, such as halides, to neutral moleculesto generate stable anionic adducts, [M + X]−, as a means ofincreasing the negative ion ESI response. Kumar et al. (2004)extended the investigation using different model compounds,including dicarboxylic acids and benzoic acids. They showedthat the collision-induced dissociation (CID) mass spectraof [M + X]− ions reflected the gas phase basicity of boththe halide ion and [M-H]− ion of the analyte. The analytesshowed a greater tendency to form adduct ions with Cl−

under ESI conditions compared with the other halide ions,

such as F−, Br−, and I−. Sheen and Her (2004) later reportedan excellent application of fluoride adduct formation (Fig-ure 31.9) for the sensitive quantitation of neutral drugs inhuman plasma. They found that the fluoride, chloride, andbromide adduct of the neutral drugs exhibited intense sig-nals in negative ion ESI. Under CID, the major product ionsof bromide and chloride adducts were the nonspecific bro-mide and chloride anions, respectively. In contrast, fluorideadducts produced strong [M-H]− ions, as well as [M-H]−

product ions with good intensity and reproducibility (Figure31.10).

Cationic adducts, such as ammonium or sodium, havebeen used as precursor ions in positive ESI mode to increaseESI responses of neutral molecules (Said et al., 2012).Amine additives have been used to suppress the forma-tion of other adduct ions and increase the ESI sensitiv-ity of the cholesterol-lowering drug, such as simvastatin,in human plasma. Zhao et al. (2002) compared the effectof mobile phases made from ammonium acetate and alkyl(methyl, ethyl, dimethyl, or trimethyl)-substituted ammo-nium acetate on the positive ESI response of simvastatinammonium adduct. When alkyl ammonium buffer was used,it was observed that simvastatin alkyl ammonium-adduct ionwas the only major molecular ion, while the formation ofother adduct ions ([M + H]+ , [M + Na]+ , and [M + K]+ )was successfully suppressed. Among the various alkyl-substituted ammonium acetate buffers, methylammoniumacetate buffer was the best one for improving the sensitivity(up to several-fold) for the simvastatin LC-MS/MS quantita-tion compared with that obtained using ammonium acetatebuffer.

CONCLUSIONS 399

0160 170 180 190 200

201

201(a)

202 221227

210 220 230 240 250m/z

%

100

0175 180 185 190 195 200 205 210 215 220 225 230 235 240 245 250 255

217

217

399

383413

179199

433

453

437 457

(b)

213

399

400

417

437

438

383413

143 145 157 165 171

179

160

199

200 219221

225 241 255

433

424445

453

454

439

457

463

458

469 477 493

221233

237243 256

m/z

m/z

%

100

0350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500

(c)

%

100

m/z0

350

360 370 380 390 400 410 420 430 440 450 460 470 480 490 500

(d)

%

100

m/z0120 130 140 150 160 170 180 190 200 210 220 230 240 250 260

(e)

%

100

FIGURE 31.9 The fluoride attachment mass spectra: (a) mephenesin, (b) guaifenesin, (c) simvas-tatin, (d) podophyllotoxin, and (e) inositol (reproduced from Sheen and Her (2004) with permissionof John Wiley & Sons, Ltd).

31.6 CONCLUSIONS

There are many challenging issues in LC-MS/MS bioanaly-sis and those challenges are different from a molecule to theother. The technologies and concepts covered in this chaptercan be implemented to resolve some challenging selectivity

and sensitivity issues in quantitative LC-MS/MS bioanalysis.DMS and MRM3 can be considered as a method of choicefor reducing high chemical background noise, eliminatingmatrix interferences and separating isomeric and metabo-lites in quantitative LC-MS/MS bioanalysis. The ionizationprocesses of any compound under ESI, APCI, or APPI are


0

(a)

m/z

%

100107

181

80 90 100 110 120123

130 140 150 160 170 180 190 200 210 220 230 240

107

181

0

(b)

m/z

%

100

187

80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240

123

197

0

(e)

m/z

%

100

80 90 100 110 120 130 140 150 160 170 180 190 200 210 220

0

(c)

m/z

%

100

260 280 300 320 340 360 380 400 420 440

283

283

383

413

179

199

354327

329

383

413

179

199

399399

0

(d)

m/z

%

100

260 280 300 320 340 360 380 400 420 440

FIGURE 31.10 The product ion mass spectra of the fluoride adducts: (a) mephenesin, (b) guaife-nesin, (c) simvastatin, (d) podophyllotoxin, and (e) inositol (reproduced from Sheen and Her (2004)with permission of John Wiley & Sons, Ltd).

highly complex. The composition of a mobile phase canhas a significant influence on the ionization efficiency ofanalyte of interest in any of the above ionization modes.Therefore, it is important to optimize the mobile phasecomposition, including additives and their concentrations,in LC-MS/MS bioanalytical method development. For some

neutral analytes, cationic or anionic adduct precursor ionscan be considered as precursor ions for desired assay selec-tivity and sensitivity. Nevertheless, it should be emphasizedthat well thought-out strategies are essential in dealing withthe issues of assay selectively and sensitivity in LC-MS/MSbioanalysis.

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