Application Note: AN 113B

High Sensitivity Methotrexate Quantitation in Human Serum withLC-MS/MS
into the clean protein precipitated human serum. All
the solvents used in this application are HPLC grade.

Quick Facts:

2.2 Mass Spectrometry Conditions

LLOQ achieved is 0.03 ng/mL with5uL injection
Liquid Chromatography: Shimadzu
Prominence UFLC
Mass Spectrometry: IONICS 3Q
Molecular Analyzer (triple quad)
ESI Source
Calibration Curve R2 = 0.99999

1. Introduction
Methotrexate is one of the oldest chemotherapy drugs
and has been in use since the late 1950’s. It was
found effective in treating various neoplastic diseases
such as acute leukemia, non-Hodgkin lymphoma andbreast carcinoma. In low doses it has also been foundeffective in the treatment of certain autoimmune
diseases. This research method presents a rapid LCMS/MS approach with high selectivity and sensitivity.
In this application note, a fast LC-MS/MS (research)
method with IONICS 3Q Molecular Analyzer wastested and verified for quantitation of methotrexate inhuman serum.

2. Method
2.1 Sample Preparation
Methotrexate was purchased from Toronto Research
Chemicals (Toronto, ON). The stock solution 25
mg/mL of Methotrexate in methanol was preparedand stored at -15°C. Human serum was purchasedfrom Sigma (Milwaukee, WI) and cleaned up byprotein precipitation by mixing one volume of humanserum, one volume of 0.1% formic acid and twovolumes of acetonitrile. The mixture was vortexed for
1 min followed by centrifugation for 15 min. The
supernatant was transferred to clean container andused as a matrix for methotrexate quantitation. A
series of methotrexate standards from 0.03 to125ng/mL were prepared by spiking the stock solution

IONICS 3Q Molecular Analyzer equipped with an ESI
source was used to perform the analysis. Instrumentconditions were:Entrance Voltage (Volts)
20
HSID Temp. (oC)300
Nebulizer Gas Setting
400
Drying Gas Setting
120
Heating Gas Setting
350
Source Temp. (oC)250
Dwell Time (ms)100
Pause Time (ms)5
Table 1 Selected MRM operating conditions formethotrexate

MRM
455.1/308.0
455.1/134.0

CE
2848

CCL2-100-100

CCL4-90-90EV
3030

2.3 LC Conditions
The separation was performed on a ShimadzuProminence UFLC system which includes binary
pumps, autosampler, degasser, column oven. Asample volume of 5 µL is injected into a Imtakt C8column (2.0 x 75 mm, 3 μm) at room temperature.
The LC time program is illustrated as Table 2. The
total LC cycle time is 3 min and liquid flow rate is 0.4mL/min.
Table 2 LC Gradient Conditions; Solvent A is 0.1%formic
acid, Solvent B is methanol with 0.1% formic acid

Time (min)
00.1
0.6
11.1
3Solvent B composition (%)
55959555

Global Headquarters: 32 Nixon RD, Unit 1, Bolton ON, L7E 1W2, Canada. Phone: 1-905-857-5665, Fax: 1-905-857-3040, www.ionics.ca
For Research Use OnlyApplication Note: AN 113B
3. Results

Figure 2 Calibration Curve for 455.1/134.0

3.1 Extracted Ion Chromatogram (EIC)The EIC for 455.1/134.0 and 455.1/308.0 for blankserum are shown in Figure 1A and 1B and the EIC
for 455.1/134.0 and 455.1/308.0 for LOQ (0.03
ng/mL in human serum) are shown in Figure 1C and1D.

Figure 3 Calibration curve for 455.1/308.0

Figure 1. (A) EIC of 455.1/134.0 in serum blank; (B) EIC of455.1/134.0 for 0.03 ng/ml in human serum; (C) EIC of
455.1/308.0 in serum blank, and (D) EIC of 455.1/308.0
for 0.03 ng/ml in human serum

3.2 Linearity

The calibration curve generated for both MRM
transitions using 1/x weighting are good and theresults for 455.1/134.0 transition (Figure 2) and
455.1/308.0 transition (Figure 3). Good linearity (R2= 0.9999) was obtained for a concentration range of0.03 to 125 ng/mL.
Table 1 Intra-day variability (%RSD)

MRM
455.1/134.0
455.1/308.0

QC1(low)
5.1
6.9

QC2(med)1.2
1.8

QC3(high)
1.0
0.5

Table 4 Inter-day variability (%RSD)

MRM
455.1/134.0
455.1/308.0

QC1(low)
2.6
7.0

QC2(med)
3.0
3.6

QC3(high)
3.5
3.8

3.3 Quantitation Results
The inter and intra-day variability for this experimentwere excellent as shown in Table 3 and 4. The intraday variability is determined by processing 4
replicates of each QC sample and the inter-dayvariability is determined with 4 replicates in 3 batches.

4. Conclusion
A fast, sensitive, and accurate LC-MS/MS method wasdeveloped for methotrexate in serum using IONICS
3Q Mass Analyzer. An LLOQ of 0.03 ng/mL (5 uLinjection) was obtained. An excellent calibration curve
for methotrexate was also obtained.

5. Contact Information
To learn more about IONICS Mass Spectrometry, ourproducts or services please visit our website orcontact us directly.

Global Headquarters: 32 Nixon RD, Unit 1, Bolton ON, L7E 1W2, Canada. Phone: 1-905-857-5665, Fax: 1-905-857-3040, www.ionics.caFor Research Use Only