Clinical Chemistry 53:10
1827–1834 (2007)
Automation and
Analytical Techniques
Methanol-Associated Matrix Effects in Electrospray
Ionization Tandem Mass Spectrometry
Thomas M. Annesley*
Background: Matrix effects can profoundly reduce the
performance of electrospray ionization mass spectrometry. Preliminary observations indicated that the methanol used in the mobile phase could be a source of
differential ionization or ion suppression.
Methods: Drug stability studies, analysis of biological
extracts, mixing experiments, and postcolumn infusions
were used to test 9 commercial methanols for ionization
differences in liquid chromatography-tandem mass
spectrometry assays for immunosuppressants. Area responses for the drugs and internal standards were
compared for mobile phases prepared with each selected methanol. Postcolumn infusion experiments were
performed to confirm the degree of ionization differences occurring at the ion source, and to evaluate the
proportions of ammonium, sodium, and potassium
adducts.
Results: The decrease in signal for the immunosuppressant drugs was shown to result from differential ionization associated with the selected methanols. Product ion
intensity varied by 10-fold among the methanols tested.
For sirolimus, tacrolimus, and mycophenolic acid, the
percentage change in ionization was the same for the
drug and its corresponding internal standard. Postcolumn sirolimus infusion evaluation revealed that a 1000fold analyte concentration difference did not affect
ionization. The proportions of ammonium, sodium, and
potassium adducts of sirolimus precursor ions differed
in relation to the source of methanol.
Conclusions: Organic solvents used in mobile phases
and extract preparation of biological samples may be
associated with ion suppression, affecting adduct formation and assay sensitivity.
© 2007 American Association for Clinical Chemistry
Department of Pathology, University of Michigan Health Sciences Center,
Ann Arbor, MI.
* Address correspondence to the author at: University Hospital, Rm.
2G332, 1500 East Medical Center Dr., Ann Arbor, MI 48109-5054. Fax 734-7634095; e-mail annesley@umich@edu.
Received April 26, 2007; accepted July 31, 2007.
Previously published online at DOI: 10.1373/clinchem.2007.090811
HPLC coupled with tandem mass spectrometry (MS/
MS)1 is being increasingly used in clinical laboratories.
Applications of liquid chromatography-tandem MS (LCMS/MS) include the quantification of testosterone, mycophenolic acid, 25-hydroxyvitamin D2 and D3, homocysteine, methylmalonic acid, acylcarnitines, plasma
metanephrine and normetanephrine, purines and pyrimidines, and antiretroviral drugs (1–9 ).
One use of LC-MS/MS at my institution is in assays for
quantification of immunosuppressants in whole blood
and serum. In sample preparation and chromatography
for these assays, we use methanol because it is readily
available and less costly than acetonitrile. The use of
methanol as a mobile phase component in MS methods
for immunosuppressants has been frequently reported
(2, 10 –14 ). In our laboratory we observed a slow loss of
32-desmethoxyrapamycin, the internal standard for sirolimus, if the 8 ␮g/L methanolic working solution was
stored at ambient temperature. This solution was very
stable if stored at ⫺20 °C, however, so we now store the
larger stock bottle at this temperature and pour off a daily
working volume. We presumed that the loss resulted
from degradation of 32-desmethoxyrapamycin in the
Fisher Optima methanol we were using, an effect similar
to that reported for the internal standard ascomycin when
dissolved in some brands or grades of acetonitrile (15 ). I
investigated whether several different sources and grades
of methanol would correct the slow loss of 32-desmethoxyrapamycin at ambient temperature, and also be
suitable for use in the mobile phase. When tested as the
solvent for preparation of the working internal standard
solution, no improvement in the slow loss of the 32desmethoxyrapamycin was found. However, when used
as the organic component of the mobile phase, I noted
large differences in the ionization of the immunosuppressants and their internal standards when different sources
and grades of methanol were evaluated.
1
Nonstandard abbreviations: MS/MS, tandem mass spectrometry; LCMS/MS, liquid chromatography-tandem MS; ESI, electrospray ionization.
1827
1828
Annesley: Methanol-Related Matrix Effects in ESI-MS
Coeluting components originating from the sample
matrix have previously been shown to negatively affect
(ion suppression) or positively affect (ion enhancement)
the analyte signal in LC-MS analyses. This report describes the phenomenon of ionization changes related to
the organic solvent used in the LC-MS/MS analysis.
Materials and Methods
reagents
Ammonium acetate (SigmaUltra) and ascomycin were
purchased from Sigma-Aldrich. Formic acid (laboratory
grade) was purchased from Fisher Scientific. Mycophenolic acid was a gift from Roche Bioscience (Palo Alto,
CA) or was purchased from Sigma-Aldrich and A.G.
Scientific. Mycophenolic acid carboxybutoxy ether was a
gift from Roche Diagnostics (Mannheim, Germany). Mycophenolic acid glucuronide was a gift from Roche Bioscience or was purchased from Analytical Services International. 32-Desmethoxyrapamycin was a gift from
Wyeth Pharmaceuticals (Pearl River, NY), tacrolimus was
a gift from Fujisawa Healthcare (Deerfield IL), and sirolimus was purchased from LC Laboratories.
commercial methanol sources
Fisher Brand Optima, Fisher Brand ACS, Fisher Brand
HPLC, Fisher Brand Optima LC/MS, EMD Omnisolv,
Baker Ultra Resi-analyzed, and Baker Analyzed LC/MS
methanol were purchased from Fisher Scientific.
Riedel-de Haen Chromasolv LC-MS methanol was purchased from Sigma-Aldrich, and Super Methanol was
purchased from American Bioanalytical. Two separate
lots were also evaluated for 7 of the 9 sources of methanol
(see Table 1 in the Data Supplement that accompanies the
online version of this article at http://www.clinchem.
org/content/vol53/issue10). My laboratory routinely
used Fisher Optima methanol at the time of the evaluation
of the different methanol types. Therefore, I chose to use
the lot number of Optima methanol in use at that time as
the reference solvent to which others were compared,
including a subsequent new lot of Optima methanol.
lc-ms/ms analyses
I quantified sirolimus and tacrolimus in whole blood as
previously described (16 ) using commercial calibrators
and controls as part of the analyses (17 ). Sirolimus was
analyzed after protein precipitation and solid-phase extraction, and tacrolimus after protein precipitation alone.
I quantified mycophenolic acid and mycophenolic acid
glucuronide as previously described (18 ) using calibrators and controls prepared in-house. Each of the immunosuppressant assays referenced above employs gradient
chromatography using 2 common mobile phases: 2
mmol/L ammonium acetate plus 1 mL/L formic acid
in water (mobile phase A) and 2 mmol/L ammonium
acetate plus 1 mL/L formic acid in methanol (mobile
phase B).
Chromatography and MS detection were performed
using an Agilent 1100 solvent delivery system and a
Waters Quattro Micro tandem mass spectrometer
equipped with a Z-spray ion source operated in the
positive electrospray ionization (ESI) mode. The instrument control software was Waters MassLynx 4.0.
comparison of peak area responses
I prepared batches of mobile phase B in 125 mL Nalgene
amber high-density polyethylene bottles to evaluate the
effect of each commercial methanol on the integrated peak
areas for the immunosuppressant drugs and internal
standards. The Nalgene bottles were rinsed with distilleddeionized water, dried, and then rinsed twice with the
selected methanol before preparation of mobile phase B.
Graduated cylinders were also rinsed with the selected
methanol before mobile phase B preparation. The same
formic acid and ammonium acetate stock solutions were
used to prepare each batch of mobile phase B, the only
variable being the brand or grade of methanol. Each
mobile phase B solution was prepared on the same day of
use.
Extracts prepared from calibrators, controls, or deidentified patient specimens (institutional review board approved) were injected into the LC-MS/MS system, and
the integrated peak areas for the drug and internal
standard were calculated using Waters MassLynx software. The concentration of drug was also quantified by
use of peak area ratios. For each series of extracts, the
Agilent 1100 system and analytical column were reequilibrated with new mobile phase B prepared with a
different commercial methanol, and then the same series
of extracts (same autosampler vials) was reanalyzed with
the new mobile phase B preparation. To verify that
differences in response for the drugs or internal standards
were not due to instrument drift or analyte degradation
during the day, on 2 occasions I used mobile phase B
preparations from methanol brands in 1 sequence to
analyze extracts, and then reversed the order and repeated the analyses with the same mobile phase preparations. Each brand or grade of methanol was also tested
again on a separate day, with a new preparation of mobile
phase B.
drug stability in commercial methanols
To determine if analyte degradation in commercial methanol contributed to the observed changes in area response, I prepared 8 ␮g/L working solutions of the
internal standards 32-desmethoxyrapamycin and ascomycin in 1 lot of each methanol. These solutions were then
used for the precipitation and solid-phase extraction of
calibrators, controls, or patient specimens containing
sirolimus and tacrolimus (16, 17 ). Fisher Optima-based
mobile phase B was used for the chromatography (16 ), so
the methanol used for the sample preparation was the
only variable. Changes in the area response for the drugs
or internal standards were compared.
Clinical Chemistry 53, No. 10, 2007
1829
postcolumn infusion
I performed postcolumn infusion studies to evaluate (a)
whether the changes in peak area related to the choice of
methanol were attributable to ionization differences in the
ion source rather than drug degradation during chromatography; (b) whether the concentration of the drug being
infused affected the ionization efficiency; and (c) if
changes occurred in the relative amounts of the ammonium, sodium, and potassium adduct ions formed during
the ionization process.
In the 1st experiment, I infused a 10 ␮g/L methanolic
sirolimus solution, using a tee, into the mass analyzer. The
syringe infusion rate was 10 ␮L/min, and the LC flow rate
was 400 ␮L/min (20% mobile phase A, 80% mobile phase
B). The signal for the m/z 931.63864.5 transition was
integrated for 30 s. The variable in this study was methanol used to prepare mobile phase B.
Heller (19 ) recently showed that the analyte:matrix
mass ratio can affect the analyte response and the analyte:
internal standard response ratio, and proposed a matrix
effect map concept to test for matrix effects in atmospheric
pressure ionization MS. To see whether varying the
analyte:methanol (i.e., matrix) ratio would highlight the
presence of ion loss resulting from the methanolic mobile
phase, I performed a 2nd experiment using postcolumn
infusions as described above, but with 4 different concentrations of sirolimus (10 mg/L, 1 mg/L, 100 ␮g/L, and 10
␮g/L). In this experiment the variable was the concentration of sirolimus tested against mobile phase B prepared
with a selected brand or grade of methanol. The m/z
931.63864.5 transition for sirolimus was monitored.
In the 3rd experiment, I infused a 10 mg/L methanolic
sirolimus solution into the mass analyzer. The infusions
were performed with 80% mobile phase B and a 400
␮L/min flow rate. During each infusion a precursor ion
scan from m/z 900 to m/z 990 was performed (30-s integration). The variables studied here were the methanol
used to prepare mobile phase B and the precursor adduct
ions formed during the ionization process.
Results
For each experiment the area response obtained for the
Optima methanol was designated as 100%, and the area
response for the other methanols was expressed as a
percentage of this reference area.
Because the same concentration of internal standard
was added to the sample before extraction and analysis, I
compared the mean area responses obtained with LCMS/MS for each commercial methanol used as the solvent
in mobile phase B. The percentage area response of the
multiple-reaction monitoring transition m/z 901.53834.4
for 32-desmethoxyrapamycin, the internal standard for
sirolimus, as a function of the methanol tested is shown in
Fig. 1. Two things are apparent from the data. First,
10-fold differences in the m/z 834.4 product ion occurred
with the various brands of methanol. Second, ion production differed between 2 lots of the same methanol type.
Fig. 1. Relative LC-MS/MS area response for 32-desmethoxyrapamycin vs methanol brand or grade.
Lot 1 of Fisher Optima methanol was designated as 100% response. Each
individual data point represents the mean of 2 separate experiments with each
brand or lot of methanol. Additionally, the results for 2 separate lot numbers of
methanol are shown. E, Fisher Optima; F, Fisher Optima LC/MS grade; 䡺, Baker
Analyzed LC/MS grade; f, Riedel-de Haen Chromasolv LC-MS; ‚, Fisher HPLC
grade; Œ, EMD Omnisolv; 〫, Fisher ACS grade; ⽧, Baker Ultra Resi-analyzed; 췦,
American Bioanalytical Super Methanol.
The results from a separate multiple-reaction monitoring
experiment are shown in Fig. 2, in which the m/z
901.53834.4 and m/z 809.43756.3 transitions for 32-desmethoxyrapamycin and ascomycin were evaluated for the
same biological extracts. The changes in ionization for
32-desmethoxyrapamycin (Fig. 2A) are of the same magnitude as those observed for ascomycin (Fig. 2B).
Although there were differences in ionization efficiency among the methanols, the ionization of analyte in
relation to internal standard changed similarly as a function of the quality of the methanol in the mobile phase, so
that the area ratios and calibration lines used to calculate
drug concentrations in patient specimens were similar
(Fig. 3, Supplemental Data Fig. 1). The 2 exceptions were
sirolimus and mycophenolic acid glucuronide when analyzed with the Baker Analyzed LC/MS methanol. The
raw product ion signals for both sirolimus and desmethoxyrapamycin were both markedly decreased, but the
signal reduction was approximately 84% for sirolimus
1830
Annesley: Methanol-Related Matrix Effects in ESI-MS
Fig. 2. Relative LC-MS/MS area response for (A) 32-desmethoxyrapamycin and (B) ascomycin vs methanol brand or grade.
Whole blood calibrators and controls containing sirolimus and tacrolimus were
supplemented with the internal standards and extracted using the protocols
referenced in Materials and Methods. Fisher Optima methanol was designated
as 100% response. E, Fisher Optima; F, Fisher Optima LC/MS grade; 䡺, Baker
Analyzed LC/MS grade; f, Riedel-de Haen Chromasolv LC-MS; ‚, Fisher HPLC
grade; Œ, EMD Omnisolv; 〫, Fisher ACS grade; ⽧, Baker Ultra Resi-analyzed; 췦,
American Bioanalytical Super Methanol.
and approximately 89% for desmethoxyrapamycin. Thus,
the area ratios for the sirolimus calibration line were
higher. Mycophenolic acid glucuronide eluted at a lower
methanol percentage and shorter retention time than the
internal standard (4.3 vs 5.8 min) during the gradient
chromatography, and the product ion signal was less
affected than the internal standard. With the Baker Analyzed LC/MS, the product ion signal for the internal
standard was also markedly decreased, a result that was
reflected by a change in the area ratios and the calibration
line.
When sirolimus was infused at concentrations spanning a 1000-fold range, the m/z 931.63864.5 transition
Fig. 3. Three-point whole blood calibration lines from whole blood extracts
quantified by LC-MS/MS using the selected methanols in the mobile phase.
(A), sirolimus. (B), tacrolimus. E, Fisher
Optima; F, Fisher Optima LC/MS grade; 䡺,
Baker Analyzed LC/MS grade; f, Riedel-de
Haen Chromasolv LC-MS; ‚, Fisher HPLC
grade; Œ, EMD Omnisolv; 〫, Fisher ACS
grade; ⽧, Baker Ultra Resi-analyzed; 췦,
American Bioanalytical Super Methanol.
yielded a proportional signal response and no apparent
change associated with the analyte:matrix ratio (Fig. 4).
The 4 methanol brands used in the comparison were
chosen because they represented the reference methanol
(Fisher Optima), a methanol brand with a modest increase
in signal response relative to the reference methanol
(EMD Omnisolv), a methanol that showed a moderate
signal decrease (Fisher ACS Grade), and a methanol that
showed a larger signal decrease (Baker Analyzed LC-MS).
The stability of sirolimus, 32-desmethoxyrapamycin,
tacrolimus, and ascomycin during the extraction of whole
blood specimens with the 9 methanols was compared. The
relative peak area response for the tacrolimus m/z
821.43768.3 transition when these methanols were used
during the extraction process (Fig. 5A) illustrates that
degradation of tacrolimus was not a problem during
contact with the methanol. However, changes in peak
area response were observed for these same blood extracts when analyzed using the same methanol in mobile
phase B of the LC-MS/MS system (Fig. 5B), indicating
that the changes in signal occurred during the ESI process.
Similar data were obtained for the other drugs and
internal standards.
To confirm that the differences in ionization were due
to methanol-associated ion decreases rather than methanol-associated ion enhancement, I performed a mixing
experiment in which I prepared a mobile phase B containing 50% Fisher Optima and 50% Baker Analyzed LC/MS
methanol. Using 32-desmethoxyrapamycin as the test
compound, the decrease in ionization for this compound
was 87% relative to a reference mobile phase B prepared
with 100% Optima methanol. This decrease in ionization
efficiency was similar to the value shown in Fig. 1 for the
Baker Analyzed LC/MS mobile phase B. This result is
consistent with an ion reduction phenomenon rather than
an ion enhancement effect.
The degree of change in ion formation appeared to
correlate with the percentage of methanol required to
elute the compound of interest. For example, when Baker
Analyzed LC/MS methanol was used to prepare mobile
phase B, a 32% decrease in ion signal was observed for
mycophenolic acid glucuronide, which eluted 1st from
1831
Clinical Chemistry 53, No. 10, 2007
Fig. 4. Relationship between infused drug concentration and MS/MS
integrated area response for sirolimus.
the reversed-phase analytical column during the gradient
chromatography at approximately 40% mobile phase B
(18 ). A 67% ion signal reduction was observed for mycophenolic acid, which eluted next at approximately 50%
mobile phase B, and a 77% ion signal reduction for
Fig. 5. Relative LC-MS/MS area response for tacrolimus when (A)
whole blood extracts were prepared with a selected brand or grade of
methanol, and (B) when the same extracts were analyzed by LCMS/MS with mobile phase B prepared from the same methanol.
Lot 1 of Fisher Optima methanol was designated as 100% response. E, Fisher
Optima; F, Fisher Optima LC/MS grade; 䡺, Baker Analyzed LC/MS grade; f,
Riedel-de Haen Chromasolv LC-MS; ‚, Fisher HPLC grade; Œ, EMD Omnisolv; 〫,
Fisher ACS grade; ⽧, Baker Ultra Resi-analyzed; 췦, American Bioanalytical Super
Methanol.
mycophenolic acid carboxybutoxy ether, which eluted at
approximately 55% mobile phase B. By comparison, the
ion reduction for desmethoxyrapamycin, which was chromatographed using an even higher percentage of mobile
phase B (17 ), averaged 87% with this commercial methanol (Fig. 1).
When 1 lot of Riedel-de Haen Chromasolv LC-MS
methanol, which had shown a mean 16% signal reduction
for 32-desmethoxyrapamycin (Fig. 1), was used, the percentage of reductions were 8% for mycophenolic acid
glucuronide, 9% for mycophenolic acid, and 10% for
mycophenolic acid carboxybutoxy ether. Although these
small changes were not statistically significant, they follow the same trend noted above.
The sirolimus adduct ion profiles for selected lots of 5
methanols used to prepare mobile phase B are shown in
Table 1. As shown by the data, the relative ratios of the
ammonium, sodium, potassium, and an as yet unidentified m/z 972.7 ion of sirolimus differed for the 5 methanols
tested. At a mobile phase composition of 80% methanol,
the sodium adducts were 130% and 114% that of the
ammonium adducts for the Baker Analyzed LC/MS and
Riedel-de Haen Chromasolv LC-MS methanols, 48% and
33% for the Fisher Optima and American Bioanalytical
methanols, and 14% for the EMD Omnisolv methanol.
Discussion
Resuts for mixing experiments, drug stability studies,
analysis of patient blood extracts, and postcolumn infusion experiments confirm that the methanol used in
LC-MS/MS can be associated with ionization changes
during ESI MS. Unidentified impurities in acetonitrile
have previously been reported to cause degradation of the
internal standard ascomycin (15 ), thereby affecting the
accuracy of results. My data show that matrix effects can
occur with the methanol in the mobile phase. Because all
of the commercial methanols I tested are reported to be
ⱖ99.8% pure, I infer that the ionization changes are
caused by trace contaminants in the solvent.
Using acetonitrile as the electrospray LC-MS solvent,
Hewavitharana and Shaw (20 ) demonstrated that the
relative intensities of the molecular ion, ammonium adduct, and sodium adduct for a model test compound,
BAY 11-7082, changed with the acetonitrile composition
in the mobile phase. In the immunosuppressant assays,
ammonium acetate is added to the mobile phase (2, 11–
14, 21, 22 ) to promote formation of the ammonium adduct, so that the molecular ion should not be observed.
My data show that the relative intensities of ammonium,
sodium, and potassium adduct ions differed with the
methanol type used in the mobile phase. The shift toward
higher percentages of sodium adducts with some methanol types (Table 1) could contribute to the lower ion
response for the ammonium precursor ion that is used in
the immunosuppressant assays. The higher percentages
of sodium adducts may result from sodium contamination or the presence of some compound that facilitates
1832
Annesley: Methanol-Related Matrix Effects in ESI-MS
Table 1. Relative proportions of sirolimus adduct ions for different methanol sources.a
m/z
m/z
m/z
m/z
931.6
936.6
952.7
972.7
[M⫹NH4]⫹
[M⫹Na]⫹
[M⫹K]⫹
(unknown)
Fisher
Optima
Baker
Analyzed
LC/MS
American Bioanalytical
Super Methanol
EMD Omnisolv
Riedel-De Haen
Chromasolv LC-MS
1
0.48
0.28
0.18
1
1.30
0.25
0.55
1
0.33
0.12
0.07
1
0.14
0.05
0.04
1
1.14
0.18
0.14
a
Results are expressed relative to the ammonium adduct precursor ion monitored in the assay. The protonated molecular ion of sirolimus does not form and
therefore was not observed or included. The syringe infusion rate was 10 ␮L/min, mobile phase 80% B, and LC flow rate 400 ␮L/min.
formation of sodium adducts. However, a shift toward
sodium adduct ion formation cannot fully explain the
losses in the intensities of the precursor and product ions.
The American Bioanalytical methanol showed no increase
in the percentage of m/z 936.6 sodium adduct ion for
sirolimus compared to the Optima methanol (Table 1), yet
ion intensities decreased for the m/z 834.4 product ion of
32-desmethoxyrapamycin (Fig. 1), the m/z 756.3 product
ion for ascomycin (Fig. 2), the m/z 864.5 product ion for
sirolimus, and the m/z 768.3 product ion for tacrolimus
(Fig. 5). In contrast, EMD Omnisolv methanol showed the
lowest percentage of nonammonium adducts, but did
have higher intensities for these same product ions. This
result highlights that the ionization of compounds in ESI
MS is affected by many unidentified subtle factors that
require further study.
In the case of sirolimus and tacrolimus, the structural
analog internal standards eluted at the same time, or very
close to the specific drug of interest (i.e., same percentage
of methanol). Thus, the percentage loss of ions was similar
for both compounds and therefore minimally affected the
peak area ratios. This similar change in ionization emphasizes the importance of using an internal standard that
behaves as closely to the compound of interest as possible
(23 ).
Ion suppression is an important phenomenon in
LC-MS analyses (24 –27 ). It is associated with endogenous
compounds in biological matrices, as well as ion-pairing
agents used to improve liquid chromatographic separation. Various protocols have been developed for evaluating the presence of ion suppression in LC-MS assays.
These include postcolumn infusion (28 ), postextraction
supplementation studies (25 ), standard line slope comparisons (26 ), and analyte:matrix ratios (19 ). In the case of
ion suppression associated with the solvent used in the
LC-MS, these protocols may not detect ion suppression
because they primarily detect ion suppression from the
biological matrices. However, there are analytical parameters that can be monitored to evaluate whether solventrelated ionization effects are occurring in LC-MS/MS.
Useful information may be gained by comparing assay
performance with several commercial sources of solvents
to determine if one solvent provides improved assay
sensitivity. A comparison of in-house assay characteristics
with those of published LC-MS/MS assays for the same
compound may help identify solvent-related ionization
effects on the limit of quantification or imprecision of the
assay.
I did not investigate what impurities are responsible
for ion suppression and alteration of the types of ions
present in the spectrum, nor the types of additional
purification steps that might alleviate the matrix effects
associated with the solvents used in chromatographic
mobile phases. These variables might be studied by
full-scan mass spectra and perhaps other analytical methods to characterize the impurity content in commercial
brands or grades of methanol, and the development of
subsequent solvent purification schemes to remove these
compounds. This type of information may further our
understanding of the fundamental chemistry and physics
of the ESI process.
Because the methanol-associated ion reduction was
greatest with the high-percentage organic solvent used to
analyze the immunosuppressant drugs, the effect may not
be as important or evident for LC-MS/MS assays that
require a lower methanol percentage for elution. Because
of the specificity of MS, however, many assays use a
simple sample cleanup and a ballistic gradient. Furthermore, newer columns such as phenyl-hexyl (29 ) and
pentafluorophenyl-propyl (30 ) are being used because
they require a higher percentage of organic solvent to
elute compounds, which purportedly provides better desolvation and increased response for compounds. Hydrophilic interaction chromatography columns (31, 32 ) are
being applied for the analysis of polar compounds. This
type of column requires a high percentage of organic
solvent in the initial mobile phase to retain compounds of
interest, which again should be associated with better
desolvation and increased response. If the mobile phase
organic solvent itself is associated with ion losses, however, then the actual analytical response in the ESI MS
may be compromised.
Although my experiments demonstrate that the methanol used in the chromatographic solvent can be associated with variations in ionization efficiency, it should be
noted that this phenomenon was evaluated with 1 type of
ion source and interface, with mobile phases containing 2
mmol/L ammonium acetate plus 1 mL/L formic acid,
and with LC-MS/MS assays that require high organic
solvent concentrations for the chromatographic portion of
Clinical Chemistry 53, No. 10, 2007
the analyses. It is possible that other source designs, or
different source operating conditions, may be less susceptible to methanol-associated ionization effects. Other variables such as decreased flow rate (ultra-performance LC,
capillary or nano-ESI) or postcolumn flow splitting may
also diminish this effect. Thus, the degree of ionization
efficiency that I observed may not be relevant or as
significant with other instruments or analytical conditions. Nonetheless, my experience shows that an assumption that any high-purity methanol will behave similarly
may not be valid, and that important information may be
gained by comparing assay performance in LC-MS/MS
with several commercial sources of solvents to determine
if one works better for the assay in question.
Grant/funding support: None declared.
Financial disclosures: None declared.
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