Everolimus-d4: An Internal Standard for Quantitation of Everolimus and Related Immunosuppressants by LCMS

By: Yunming Ying, Huahua Jian, Isil Dilek, Uma Sreenivasan,

Everloimus-d4 was synthesized and certified for use as an internal standard in LCMS applications. Use of the standard in LCMS applications was demonstrated by quantitation of an everolimus control sample against a calibration curve.


The synthesis was developed from rapamycin by analogy to published procedures for native Everolimus.1


Ethylene-d4 glycol was monosilylated with t-butyldimethylsilylchloride in the presence of base and converted to the trfilate (1) with triflic anhydride/2,6-lutidine.1a Rapamycin was alkylated with 1 in 2,6-lutidine in toluene to give TBDMS protected everoliums-d4 followed by deprotection with 1N HCl to form the product everolimus-d4.1b,c

Certification of Everolimus-d4

Everolimus-d4 was certified for use as a reference material by testing for chromatographic purity, isotopic purity and residual impurities (Table 1). The product is suitable for use as internal standard for analysis of everolimus and other immunosuppressants by mass spectrometry.

Use of Everolimus-d4 in LCMS Applications

Everolimus-d4 was used as internal standard to quantitate the concentration of an independently prepared control sample of native everolimus to an everolimus calibration curve. Both native and labeled everolimus were screened on triple quadruple and QTOF instruments. MSMS of Everolimus- d4 was performed by infusing to Agilent 6410 QQQ (FR=135V, CE= 65V). The transitions observed for everolimus- d4 were 984.6→393.3, 409.3 and 655.4; corresponding to native everolimus 980.6→389.3, 409.3 and 651.4.

Quantitation was performed on a Waters Xevo-G2 QTOF in TOF MS mode. Ions monitored are M+Na+ (m/z 980.5706 and 984.5958 for native and labeled respectively). The extraction window was ± 0.010 Da of theoretical exact mass.

Internal standard Spiking Solution: Everolimus-d4 was prepared at 5 μg/mL in methanol.
Everolimus Control sample: Everolimus sample was prepared at 100 μg/mL in acetonitrile. Native everolimus was procured from Sigma.
Everolimus Calibration Curve: A four point calibration curve of everolimus was prepared with points from 54 to 135 μg/mL in acetonitrile.
Working solutions: Samples were prepared for analysis by adding 1000 μg internal standard solution into 50 μg of each sample and curve point and 700 μL of methanol. The working concentration was 2-3 μg/mL.

Chromatograms and method details are provided in Figures 1&2 and Tables 2&3. Spectra of triple quadruple product ion scan are provided in Figure 4.


The internal standard proved suitable for use in quantitative applications. The calibration curve was linear with r2=0.9979. Concentration of the control sample was 106.27 μg/mL with 1.54 %RSD.

Table 1: Certification of Everolimus--d4


Analytical Test Method Results
Chromatographic Purity by HPLC/PDA Analysis SP10-0102 99.3%
Identity by LC/MS Analysis SP10-0107 Consistent with Structure
Isotopic Purity by LC/MS SIM Analysis
0.00% D0 vs D4
0.00% D0 2.51% D3
0.03% D1 96.70% D4
0.76% D2  
Identity by 1H-NMR Analysis USP <761>, SP10-0116 Consistent with Structure
Residual Solvent Analysis by GC/FID Headspace AM1087 1 4.91%
Residual Water Analysis by Karl Fischer Coulometry USP <921>, SP10-0103 0.46%
Purity Factor2   94.0%

1Validated analytical method

2 Purity Factor = (100 - wt% residual solvent - wt% residual water) x Chromatographic Purity/100

Extracted Ion Chromatogram of Everolimus

Figure 1: Extracted Ion Chromatogram of Everolimus and Everolimus-d4

Calibration Curve of Native Everolimus

Figure 2: Calibration Curve of Native Everolimus

Compound name: Everolimus
Correlation coefficient: r = 0.998989, r^2 = 0.997979
Calibration curve: 0.944453*  x + 2.48711
Response type: Internal Std (Ref 2), Area* (IS Conc./ IS Area)
Curve type: Linear, Origin: Exclude, Weighting: Null, Axis trans : None

Table 2: LC Conditions

Column Waters Xselect CSH C18 3.5uM, 2.1*10mm Guard Column
Column Temperature 35.0 C
Solvent A Water with 0.1% formic acid
Solvent B Methanol with 0.1% formic acid
Flow Rate 0.400 mL/min
Injection Volume 5 μL with needle wash
Gradient Time(min) %A %B Curve
Initial 30 70  
0.2 30 70 6
0.6 0.1 99.9 6
1.0 0.1 99.9 6
1.2 30 70 6
5 30 70 6


Table 3: MS Detection Conditions

Acquisition mass range: 400 Da to 1500 Da.
Calibration mass range: 430.995 Da to 1450.119 Da
Analyser Sensitivity Mode
Ion Source ES+
Capillary 3.5 kV
Sampling Cone 90 V
Extraction Cone 4 V
Source Temperature 130°C
Desolvation Temperature 450°C
Cone Gas Flow 10.0 L/Hr
Desolvation Gas Flow 1200.0 L/Hr
Collision Energy 6 V
Lock Mass 556.277100 (LeuEnk Positive MS)
Scan Time 0.300 sec
Interscan Time 0.014 sec
Data Format Centroid
Scans to Average 3.0


MSMS of Everolimus

Figure 4: MSMS of Everolimus- d4

Instrument: Agilent 6410 Triplequad.




  1. a) J. Org. Chem. 1986, 51, 3390-3391; b) US5665772; c)US2010/0094408.


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