High Sensitivity Methotrexate Quantitation in Human Serum utilizing LC-MS/MSSha Joshua Ye; George Scott
IONICS Mass Spectrometry, Bolton, Ontario, CanadaIntroduction
Methotrexate is one of the oldest chemotherapy drugs and has been in use since the late1950’s. It was found effective in treating various neoplastic diseases such as acute leukemia,
non-Hodgkin’s lymphoma and breast carcinoma. In low doses it has also been found effectivein the treatment of certain autoimmune diseases. However, when taken in overdose,
methotrexate may cause a variety of health issues such as renal or gastrointestinal damage.
In order to avoid these potentially serious adverse effects, an accurate quantification method
is needed.
This research method presents a rapid LC-MS/MS approach utilizing IONICS’ 3Q 210
Molecular Analyzer. The method provides high selectivity and sensitivity, which was tested andverified for the quantitation of methotrexate in human serum.

MethodSample Preparation: Methotrexate was purchased from Toronto Research Chemicals(Toronto, ON). The stock solution, 25 mg/mL of methotrexate in methanol was prepared andstored at -15°C. Human serum was purchased from Sigma (Milwaukee, WI) and cleaned up
using protein precipitation by mixing one volume of human serum, one volume of 0.1% formic
acid and two volumes of acetonitrile. The mixture was vortexed for 1 min followed by centrifugation for 15 min. The supernatant was transferred to a clean container and used as a matrix formethotrexate quantitation. A series of methotrexate standards from 0.03 to 125ng/mL were prepared by spiking the stock solution into the clean protein precipitated human serum. All the solvents used in this application are HPLC grade.

LC Conditions: The separation was performed on a Shimadzu Prominence UFLC system
which includes binary pumps, an autosampler, degasser, and a column oven. A sample
volume of 5 µL was injected into a Imtakt C8 column (2.0 x 75 mm, 3μ) at room temperature.
The LC time program is illustrated in Table 3. The total LC cycle time was 3 min with a liquidflow rate of 0.4 mL/min.
Table 3: LC Gradient Conditions; Solvent A is H20 with 0.1% formic acid, Solvent B ismethanol with 0.1% formic acid
Time (min)00.10.611.1
3Solvent B composition (%)
55959555Linearity
The calibration curves generated for both MRM transitions using 1/x weighting are goodand the results for 455.1/134.0 transition (Figure 2a) and 455.1/308.0 transition (Figure2b). Good linearity (R2 = 0.9999) was obtained for a concentration range of 0.03 to 125
ng/mL.
Table 4 and Table 5 show the intra and inter-day variability for the results. The intra-day
variability is determined by processing 4 replicates of each QC sample and the inter-day
variability is determined with 4 replicates in 3 batches.
Table 4: Intra-Day Variability (%RSD)
MRM
455.1/134.0

QC1(low)
5.1

QC2(med)
1.2

QC3(high)
1.0

455.1/308.0

6.9

1.8

0.5

Table 5: Inter-Day Variability (%RSD)

Results

MRM
455.1/134.0

QC1(low)
2.6

QC2(med)
3.0

QC3(high)
3.5

Extracted Ion Chromatograms

455.1/308.0

7.0

3.6

3.8

Figure (1A): The EIC for 455.1/134.0 Serum Blank (1B) EIC of 455.1/134.0 for 0.03 ng/mL
in Human Serum. (1C) EIC of 455.1/308.0 Serum Blank, (1D) EIC of 455.1/308.0 for 0.03
ng/mL in Human Serum

Figure 2a: Calibration Curve for 455.1/134.0

Figure 2b: Calibration curve for 455.1/308.0

Mass Spectrometry Conditions: An IONICS 3Q 210 equipped with an ESI source was used
to perform the analysis. Instrument conditions were: Electrospray Voltage: 5000V, HSID
Temperature: 300oC, Nebulizer Gas Setting: 400, Drying Gas Setting: 120, Heating Gas Setting:350, Source Temperature: 250oC, Dwell Time: 100 ms, Pause Time: 5 ms.

A

This method measured MRM transitions 455.1/308.0 and 455.1/134.0, Table 2 provides abrief overview of the settings utilized while monitoring these transitions

BTable 2: Monitored MRM Transitions and Associated Instrument Settings
MRMCECCL2CCL4

EV455.1/308.0

28-100-9030

455.1/134.0

48-100-9030Conclusion
A fast, sensitive, and accurate LC-MS/MS method was developed for methotrexate inserum using IONICS 3Q 210. An LLOQ of 0.03 ng/mL (5 μL injection) was obtained.
An excellent calibration curve with R2=0.9999 for methotrexate was also obtained. Thesample preparation for this LC-MS/MS method is simple and well suited for routine
clinical analysis of methotrexate.

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