NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

 

CYCLOPHOSPHAMIDE, 4-KETOCYCLOPHOSPHAMIDE and IFOSFAMIDE in urine 8327

Formula: 1. Cyclophosphamide, C7H15Cl2N2O2P MW: 1. 261.1 CAS: 1. 50-18-0 RTECS: 1. RP5950000

 2. 4-Ketocyclophosphamide,C7H13Cl2N2O3P 2. 275.1 2. 27046-19-1 2. none

 3. Ifosfamide, C7H15Cl2N2O2P 3. 261.1 3. 3778-73-2 3. RP6050000

METHOD: 8327, Issue 1 EVALUATION: FULL Issue 1: 5 March 2021

 OSHA: None

 NIOSH REL: None

Because data on exposure limits and guidelines may change over time, 

NIOSH recommends referring to the following sources for updated 

limits and guidelines [1-3].

PROPERTIES: 1. white, crystalline powder; MP 41-45 °C or 47-49 

°C or 49.5-53 °C [4,5] (Cyclophosphamide is often purchased as the 

monohydrate, CAS 6055-19-2. These various melting points reflect 

both the compounds.) 2. white solid; MP 139-141 °C [6] 3. white 

crystalline powder, MP 39-41 °C [4,5]

 SYNONYMS: 1. N,N-bis(2-chloroethyl)-2H-1,3,2-oxazaphosphorin-2-amine-2-oxide or 2-(bis(2-chloroethyl)amino)tetrahydro-2H- 

 1,3,2-oxazaphosphorine-2-oxide.; 2. 4-oxycyclophosphamide or 2-(bis(2-chloroethyl)amino)tetrahydro-4H-1,3,2-oxazaphosphorin-4- 

 one 2-oxide.; 3. N,3-bis-(2-chloroethylamino)tetrahydro-2H-1,3,2-oxazaphosphorine-2-oxide or (2-chloroethylamino)-N-(2

 chloroethyl)tetrahydro-2H-1,3,2-oxazaphosphorine-2-oxide. 

SAMPLING

SPECIMEN: Urine

VOLUME: At least 8 mL of sample

PRESERVATIVE: None

SHIPMENT: Frozen

SAMPLE

STABILITY: Not established. Appears to be stable in

Frozen urine for long periods. See [7] for 

more details.

CONTROLS: Urine samples obtained from nonexposed individuals

ACCURACY [7]

RANGE 

STUDIED: 1 to 20 ng/mL for CP and IF, 25 to 525 ng/mL for

 4-keto-CP 

BIAS: None established

OVERALL

PRECISION (SS�rrrr): See Tables 1 and 2

ACCURACY: 103% recovery for cyclophosphamide and 4-

ketocyclophosphamide, 102% recovery for 

ifosfamide. The precision by relative 

standard deviation was no larger than 8.4% 

at any concentration level (see Tables 1 and 

2).

MEASUREMENT

TECHNIQUE: HIGH-PERFORMANCE LIQUID 

CHROMATOGRAPHY - TANDEM MASS 

SPECTROMETRY (HPLC/MS/MS)

ANALYTES: Cyclophosphamide (CP), 4-

ketocyclophosphamide (4-keto-CP) and 

ifosfamide (IF)

EXTRACTION: Liquid-liquid extraction with ethyl acetate 

COLUMN: C18 (150 X 3 mm, 3.5 μm).

MOBILE PHASES: A = 15/85/0.1% (v/v/v) 

 acetonitrile/water/acetic acid.

 B = 75/25/0.1% (v/v/v) 

 acetonitrile/water/acetic acid.

FLOW RATE: 0.3 mL/min

GRADIENT: Time (min) Mobile Phase

 Composition

0 to 2 0%B

2 to 10 0 to 30% B

10 to 18 30 to 100% B

18 to 25 100% B

25 to 27 100 to 0% B

27 to 35 0% B

INJECTION VOLUME: 10 μL

IONIZATION SOURCE: Electrospray at 3000 Volts and

 positive scan mode, nebulizer gas at

 35 psi and 10 L/min.

DETECTOR (MS/MS): Dwell time = 200 msec

 Fragmentor at 140 Volts

 Collision energy at 25 Volts.

 Nitrogen collision gas.

CALIBRATION: Standards from CP, 4-keto-CP and IF 

spiked urine solutions with internal 

standard.

RANGE: 0.5 to 25 ng/mL CP and IF; 10 to 625 ng/mL 

4-keto-CP

ESTIMATED LOD: By IUPAC definition [8]: CP and IF = 

approximately 0.1 ng/mL, 4-keto-CP = 

approximately 1 ng/mL. LOD is dependent 

upon column and instrumentation. By 

lowest standard levels: CP and IF = 0.5 

ng/mL, 4-keto-CP = 10 ng/mL.

PRECISION (SS�rr): See Tables 1 and 2.
Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine: METHOD 8327, Issue #1, 5 March 2021 - Page 2 of 10

NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

APPLICABILITY: CP and IF are drugs used to treat cancer; 4-keto-CP is a metabolite and biomarker for CP exposure. This method 

measures the quantity of the three target analytes in urine.

INTERFERENCES: None found or identified.

OTHER METHODS: This method is based upon that described by B’Hymer and Cheever [9]. A review article of LC/MS methods for 

urinary analysis of anticancer drugs may be found to be useful [10].
Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine: METHOD 8327, Issue #1, 5 March 2021 - Page 3 of 10

NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

REAGENTS:

1. Cyclophosphamide (CP) monohydrate 

reference standard.* Prepare a 0.20 

mg/mL stock solution in acetonitrile. 

Store in a refrigerator.

2. Ifosfamide (IF) reference standard.* 

Prepare a 0.20 mg/mL stock solution in 

acetonitrile. Store in a refrigerator.

3. 4-Ketocyclophosphamide (4-keto-CP) 

reference standard.* Prepare a 0.10 

mg/mL stock solution in acetonitrile. 

Store in a refrigerator. 

4. Deuterated cyclophosphamide-d6 (d6-

CP) reference standard.* Prepare a 0.15 

mg/mL stock solution in acetonitrile. 

Store in a refrigerator.

5. Internal standard spiking solution, d6-CP 

stock solution diluted to approximately 

48 ng/mL in water.

6. Acetonitrile, HPLC grade or better.*

7. Acetic Acid, glacial, LC/MS grade or 

better.*

8. Ethyl Acetate, HPLC grade or better.*

9. Acetonitrile/water (50/50, v/v) injector 

rinse solution.

10. Mobile phase A (15/85/0.1, v/v/v) 

acetonitrile/water/acetic acid. 

11. Mobile phase B (75/25/0.1, v/v/v) 

acetonitrile/water/acetic acid.

12. Potassium hydroxide solution, 5 M. 

Purchase or prepare by dissolving 28.05 

g KOH in 100 mL water.

13. Potassium phosphate buffer, 2 M (pH 

7.0). Prepare by diluting 23.0 g H3PO4 

(85%) to 100 mL with water. pH to 7.0 

using 5 M KOH solution.

14. Potassium phosphate buffer, 0.02 M (pH 

7.0). Prepare by diluting 2 M solution 

1:100.

15. Water, doubly deionized.

16. Water, HPLC grade or better for use as 

mobile phase.

*See SPECIAL PRECAUTIONS.

EQUIPMENT:

1. High-Performance Liquid 

Chromatograph equipped with a 

tandem mass spectrometric detector

2. C18 HPLC column (150 X 3mm, 3.5 μm 

particle size)

3. Autosampler capable of maintaining 7 

°C

4. Analytical balance

5. Rotary vacuum concentrator with cold 

trap and vacuum pump or nitrogen 

evaporator

6. Automatic pipettor with disposable 

tips in the 1000 μL volume delivery 

range

7. Test tube vortex mixer

8. Re-pipettor to dispense 5 mL volumes 

of liquid

9. Disposable screw-top culture tubes (16 

X 150 mm) with PFTE-lined caps

10. Metal spatula

11. Volumetric flasks; 10, 50, 100 and 200 

mL, glass

12. Autosampler vials, amber with caps, 

septa and 100 μL deactivated glass 

inserts

13. Disposable glass Pasteur pipets

14. Sintered glass filtering apparatus and 

1-L side-armed Erlenmeyer flask for 

filtration of mobile phases

15. Filters, glass microfiber, size to fit 

filtering apparatus, 0.7 μm

16. pH meter

 
Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine: METHOD 8327, Issue #1, 5 March 2021 - Page 4 of 10

NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

SPECIAL PRECAUTIONS: Wear appropriate gloves (nitrile, polyurethane, neoprene, or latex), lab coat, 

and safety glasses while handling all chemicals and human urine products. Standard precautions [11] 

should always be used when handling bodily fluids and/or extracts of bodily fluids.

Acetic acid, acetonitrile, and ethyl acetate are flammable; handle with care and use in a chemical fume 

hood. CP and IF are known carcinogens. 4-keto-CP, and d6-CP are unable to be classified, but observe all 

the required safety precautions. Reagents with manufacturer expiration dates should be observed.

SAMPLING:

1. Collect the urine in a polypropylene tube or bottle and cap.

2. Ship the sample stored in either wet or dry ice in an insulated container, store frozen upon arrival at 

the laboratory 

SAMPLE PREPARATION:

3. Thaw the urine sample to room temperature.

4. Mix thoroughly to ensure urine homogeneity.

5. Transfer 4.0 mL of urine into a 16 X 150 mm screw-capped culture tube. 

6. Add 0.5 mL of 2 M potassium phosphate buffer solution.

7. Add 0.25 mL of the 48 ng/mL internal standard spiking solution.

8. Add 0.25 mL of deionized water.

9. Ethyl Acetate Extraction. Dispense 5 mL of ethyl acetate into culture tube, place cap on culture tube, 

and vortex for 1 minute. Inversion of the tubes may be substituted if vortexing causes too much

emulsion to form (>1/2 inch or if it starts to affect the results).

10. Allow layers to separate, collect the ethyl acetate layer. If emulsions form, centrifugation of the 

samples may be performed to aid the layers in separating.

11. Repeat the extraction (steps 9-10) two more times. Collect and combine all ethyl acetate extracts using 

a Pasteur pipet.

12. Evaporate the combined ethyl acetate extract for each sample to dryness using a rotary concentrator 

or nitrogen evaporator at 30°C temperature. 

NOTE: If storing the dry extract before analysis, it is advised to store cold, in either a refrigerator or 

freezer (≤4°C).

13. Dissolve the extract in 0.25 mL of HPLC grade water. Transfer the sample into the HPLC autosampler 

vial containing the 100 μL insert and analyze per steps 24-28.

CALIBRATION AND QUALITY CONTROL

14. Prepare a CP/IF standard mixture solution by combining 1.0 mL of each stock solution and diluting to 

50 mL with 0.02 M phosphate buffer to make a 4 μg/mL concentration for each analyte.

15. Prepare CP/IF/4-keto-CP spiking solutions. The 4 μg/mL CP/IF standard mixture (from Step 14) and the 

0.1 mg/mL 4-keto-CP stock solution are diluted in 0.02 M phosphate buffer to make 8, 16, 32, 160, 320 

and 400 ng/mL CP/IF and 0.2, 0.4, 0.8, 4, 8 and 10 μg/mL 4-keto-CP solutions for spiking. Prepare one 

additional 160 ng/mL 4-keto-CP solution for the low level standard spike. 

16. Transfer 4.0 mL of non-exposed urine into a 16 X 150 mm screw-capped culture tube.

17. Add 0.5 mL of 2 M potassium phosphate buffer solution.

18. Add 0.25 mL of the 48 ng/mL internal standard (IS) solution.

19. Add 0.25 mL of the spiking solution described in step 15 to make urine samples equivalent to 0.5, 1, 2, 

10, 20, 25, ng/mL of CP/IF and 10, 12.5, 25, 50, 250, 500 and 625 ng/mL of 4-keto-CP in the original 4.0 

mL volume of urine. Blank urine that has none of the analytes is used for these standards (see step 21).

20. Process the spiked standard urine samples the same as described in the preceding Sample Preparation 

section using steps 9 through 13.
Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine: METHOD 8327, Issue #1, 5 March 2021 - Page 5 of 10

NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

21. Prepare and process one blank urine without analyte spikes to verify the source of blank urine 

contained no detectable quantity of CP, IF, or 4-keto-CP.

22. Prepare and process at least one quality control (QC) standard of CP/IF/4-keto-CP fortified urine 

using separately weighed and prepared stock solutions. QC samples should be greater than 5% of 

the sample set. QC values should normally be within ±20% of the spiked values. If not, the batch is 

considered out of control, the batch data discarded, and corrective actions taken before more 

specimens are analyzed per steps 24-28.

23. Measure the peak areas of the analytes and the IS in the chromatograms. Divide the peak area of the 

analytes by the peak area of the deuterated IS (d6-CP). Prepare calibration curves of this quotient 

versus the concentration of the standards for the three analytes. The standard curve should have a 

coefficient of determination (r2

) of equal to or greater than 0.98 to be acceptable for use. 

Furthermore, when each standard is inserted into the calibration equation, the value should be 

within ±20% of the expected value.

MEASUREMENT:

24. Set the high-performance liquid chromatograph according to the manufacturer’s recommendations 

and to the conditions listed previously.

25. Set the mass spectrometric detector to MULTIPLE REACTION MODE (MRM). Quantification mass 

transitions are as follows: CP = m/z 261 → 140, 4-keto-CP = 275 → 106, IF = 261 → 154, d6-CP = 267 

→ 140. Program MRM according to the manufacturer’s recommendations. For an Agilent model 

6410A, please see the additional conditions listed below:

MS/MS conditions using positive electrospray ionization

Analyte Precursor 

Ion

MS1

Resolution

Product

Ion

MS2

Resolution

Dwell

Time (msec)

Fragmentor

Voltage

Collision

Energy

(volt)

d6-CP 267 wide 140 unit 200 140 25

CP 261 wide 140 unit 200 140 25

IF 261 wide 154 unit 200 140 25

4-keto-CP 275 wide 106 unit 200 140 25

26. Inject 10 μL of each sample extract, calibration standard, QC, and blank.

27. Rinse the injection needle with the 50/50 acetonitrile/water solution after every injection to prevent 

carryover.

28. Measure the peak areas of the three analytes (CP, IF and 4-keto-CP) and that for the IS in the 

chromatograms. Divide the peak area of the analytes by the peak area from the IS. A representative 

chromatogram of the three analytes of interest is shown below (Figure 1). The chromatogram does 

not show the IS, which elutes at nearly the same retention time (13.4 min) as the non-deuterated CP.
Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine: METHOD 8327, Issue #1, 5 March 2021 - Page 6 of 10

NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

Figure 1. Chromatograms of urine fortified with 50 ng/mL 4-keto-CP (8.2 min), 2 ng/mL IF (12.9 min), 

and 2 ng/mL CP (13.6 min).

CALCULATIONS:

29. Determine the concentration of the three analytes in the extracts from the original urine (4.0 mL 

sample) from the curves obtained in step 23. The results can be expressed as ng/mL of each analyte 

in urine.

EVALUATION OF METHOD:

This method was evaluated and described in detail by B’Hymer and Cheever [9] and in the method backup 

data report [7]. A general summary of this previously published information is given below. 

Accuracy and Precision. Two recovery studies using two columns from different lots performed over 

several days demonstrated the accuracy and precision of this test method. The two recovery studies 

generated a total of 47 spiked urine samples at 1, 2, 4, and 15 ng/mL CP/IF levels and 25, 50,100 and 375 

ng/mL 4-keto-CP levels. The first recovery study used urine from volunteers that was pooled after 

collection and then fortified at the various levels. The study was performed over three separate 

experimental batch runs and these data are presented in Table 1. Average recoveries were between 97 and 

105% for the three analytes over the concentration levels investigated. For each batch run, the 

experimental trial consisted of three samples at three different concentration levels. The recovery for each 

level (n=9 samples) is displayed in Table 1. The second recovery study used fortified urine samples from 20 

non-exposed volunteers and demonstrated that the procedure was accurate and precise (Table 2). No 

interferences were detected in the unspiked urine from the 20 volunteer samples. Precision expressed as 

percent relative standard deviation (% RSD) was as high as 8.4% on the volunteer recovery samples (n=20). 

Overall recoveries had mean values of 102% (n=47) for IF and 103% (n=47) for both CP and 4-keto-CP; thus, 

there was no apparent bias.

Linearity. All calibration curves used during the development of this method were linear and had 

coefficients of determination of 0.99 and greater. The concentration range was 0.5 to 25 ng/mL CP and IF 

with a 4.0 mL urine sample size. The concentration range was 10 to 625 ng/mL 4-keto-CP in urine. 

Calibration curves were run at the beginning and end of all sample batch runs; calibration curve slope drift 

was found to be minimal during the development and validation of this method.
Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine: METHOD 8327, Issue #1, 5 March 2021 - Page 7 of 10

NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

Specificity. The optimized chromatographic conditions developed for this method, along with the tandem 

mass spectrometric detector, proved to be specific and have no major interferences. The mass transition 

ions of CP and IF chosen in this method had the greatest response. This strategy gave the best sensitivity 

for those two analytes. The mass transition for 4-keto-CP, m/z 275 → 106, was chosen because it was more 

unique for the compound compared to the other possible metabolites of IF, which had similar 

chromatographic retention times. This method does not use qualifying ions as it is believed that retention 

times coupled with the multiple reaction mode offer enough specificity. Qualifying ions were investigated 

during the development of the method. Those transitions and some discussion can be found in the 

method backup data report [7].

Robustness. Two Agilent Rx-C18 HPLC columns of different manufacturing lots were used during the 

recovery studies. Accuracy and precision did not appear to be affected; therefore, the method appears to 

be reproducible with any functioning Agilent Rx-C18 HPLC column. Recovery results from individual 

volunteer urine samples spiked with the three analytes indicate that the method is accurate and not 

significantly affected by individual urine sample matrix differences during analyte extraction.

Stability. Sample stability was evaluated. A five-day stability study was conducted on the final 

chromatographic sample solution. CP, IF, and 4-keto-CP appeared to be stable at 7 °C (the autosampler 

temperature) and at room temperature in laboratory light when stored in amber autosampler vials.

Range. This method should be considered accurate for the estimation of CP and IF in human urine within 

the 1 to 25 ng/mL range. It also should be considered accurate for the estimation of 4-keto-CP in human 

urine within the 25 to 525 ng/mL range. Field samples at higher levels can be diluted to a concentration 

within those ranges for analysis.

REFERENCES:

[1] ACGIH [2019]. TLVs and BEIs based on the documentation of the Threshold Limit Values for chemical 

substances and physical agents and Biological Exposure Indices. Cincinnati, Ohio: American 

Conference of Governmental Industrial Hygienists, www.acgih.org.

[2] Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area 

[2018]. List of MAK and BAT values 2018; Report 54. DFG: Deutsche Forschungsgemeinschaft, 

http://onlinelibrary.wiley.com/book/10.1002/9783527818402.

[3] SUVA (Swiss National Accident Insurance Fund) [2018]. www.suva.ch/en/material/Factsheet/occupational-exposure-limits-basics-and-application.

[4] The Merck Index [2013]. The Merck Index, 15th Edition. The Royal Society of Chemistry: Cambridge, 

UK.

[5] Pubchem [2019]. www.pubchem.ncbi.nlm.nih.gov (Cyclophosphamide and ifosfamide located 

separately. Information in the Experimental Properties sections.)

[6] Struck RF, Kirk MC, Mellett LB, el Dareer S, Hill DL [1971]. Urinary metabolites of the antitumor agent 

cyclophosphamide. Mol Pharmacol 7(5):519-529.

[7] B’Hymer C [2010]. Backup data report for NIOSH 8327: cyclophosphamide, 4-ketocyclophosphamide 

and ifosfamide in urine.

[8] IUPAC [2014]. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled by A. D. 

McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997). XML on-line corrected 
Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine: METHOD 8327, Issue #1, 5 March 2021 - Page 8 of 10

NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

version: http://goldbook.iupac.org (2006-) created by M. Nic, J. Jirat, B. Kosata; updates compiled by 

A. Jenkins. ISBN 0-9678550-9-8, https://doi.org/10.1351/goldbook.L03540.

[9] B’Hymer C, Cheever KL [2010]. Evaluation of a procedure for the simultaneous quantification of 4-

ketocyclophosphamide, cyclophosphamide and ifosfamide in human urine. J Chromatogr Sci 

48:328-333. 

[10] Mathias PI, Connor TH, B’Hymer C [2017]. A review of high performance liquid chromatographicmass spectrometric urinary methods for anticancer drug exposure of health care workers. J 

Chromatogr B 1060:316-324.

[11] CDC [2007]. 2007 Guidelines for isolation precautions: preventing transmission of infectious agents 

in healthcare settings, http://www.cdc.gov/hicpac/2007IP/2007isolationprecautions.html.

METHOD WRITTEN (REVISED) BY:

Clayton B'Hymer, PhD, NIOSH/HELD. Final editing by Dale A. Shoemaker, PhD, NIOSH/HELD.

DISCLAIMER

Mention of any company or product does not constitute endorsement by the National Institute for 

Occupational Safety and Health, Centers for Disease Control and Prevention. In addition, citations to 

websites external to NIOSH do not constitute NIOSH endorsement of the sponsoring organizations or their 

programs or products. Furthermore, NIOSH is not responsible for the content of these websites. All web 

addresses referenced in this document were accessible as of the publication date. 
Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine: METHOD 8327, Issue #1, 5 March 2021 - Page 9 of 10

NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

TABLE 1

Multiple Level Recovery Experiment of 4-Keto-CP, CP and IF

Analyte/Nominal conc.

(ng/mL)

Mean measured 

conc. (n=9)

(ng/mL)

Average percent 

recovery

(Accuracy)

Standard 

deviation

(ng/mL)

%RSDA

4-Keto-CP

25 24.2 97 1.2 5.1

100 105 105 3.4 3.2

375 386 103 13 3.4

CP

1 1.05 105 0.056 5.3

4 4.10 103 0.094 2.3

15 15.2 101 0.35 2.3

IF

1 1.00 100 0.079 7.9

4 4.08 102 0.085 2.1

15 15.2 101 0.37 2.4

Notes: Three different spiked samples were prepared at each level and analyzed on three separate 

experimental trial runs (a total of nine samples at each spike level were analyzed). The same LC column was 

used for experimental batch trials 1 and 2; a second LC column was used on trial run 3. A %RSD: percent relative standard deviation.
Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine: METHOD 8327, Issue #1, 5 March 2021 - Page 10 of 10

NIOSH Manual of Analytical Methods (NMAM), Fifth Edition

TABLE 2

Recovery of 4-Keto-CP, CP and IF Spikes from Urine Samples of 20 Non-exposed Volunteers

Analyte mean measured 

conc. (ng/mL)

Average percent 

recovery (n=20)

(Accuracy)

Measured conc. range

(low to high)

(ng/mL)

%RSDA

 4-Keto-CP

53.7 105 44.3 – 60.3 8.4

CP

2.08 104 1.97 – 2.14 2.6

IF

2.14 103 1.80 – 2.59 7.2

Notes: The prepared theoretical concentrations were 51.0 ng/mL for 4-Keto-CP, 2.00 ng/mL for CP, and 2.08 

ng/mL for IF. 

A %RSD: percent relative standard deviation.