NIOSH Manual of Analytical Methods (NMAM) 5th Edition 

BACKUP DATA REPORT

NIOSH Method No. 8327

Title: Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine

Analyte: Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide 

Author/developer: Clayton B’Hymer

Date: March 5, 2021

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.
Backup Data Report for NMAM 8327: Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine

dated 5 March 2021 - Page 2 of 16

NIOSH Manual of Analytical Methods (NMAM) 5th Edition

BACKUP DATA REPORT

Method No. 8327

Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine

Clayton B’Hymer

Substance(s): 1. Cyclophosphamide

 2. 4-Ketocyclophosphamide

 3. Ifosfamide

Exposure Limits: Not applicable

Chemical Used for Evaluation:

1. Cyclophosphamide monohydrate from Sigma Chemical, Inc., assay 97 to 103%, Lot no. 113K1406.

2. 4-Ketocyclophosphamide from Niomech IIT, GmbH, 98%, Lot no. D-18845.

3. Ifosfamide from American Pharmaceutical Partners (APP), Inc., 98%, Lot no. 200969.

GENERAL

Synopsis

Cyclophosphamide (CP) and ifosfamide (IF) are two commonly used drugs for the treatment of 

cancer. The two drugs are widely used by healthcare workers associated with cancer patients including 

nurses and pharmacy personnel who prepare dosages. The metabolism of these drugs is complex, each 

resulting in more than a dozen active and inactive metabolites [1, 2]. Some of these metabolites are 

unstable in urine [3] and therefore unsuitable for biomonitoring. The described procedure was developed 

for the detection and quantification of the two parent drugs and one of the inactive metabolites, 4-

ketocyclophosphamide (4-keto-CP) in urine. It is hoped that this metabolite will serve as a good marker 

for exposure. Urine from an exposed population is collected, shipped cold, and stored frozen at -70 to -80

°C. The samples are thawed for analysis. Liquid-liquid extraction (LLE) using ethyl acetate collects the 

three analytes from the urine samples. The ethyl acetate extracts are evaporated and the dry residues 

are dissolved in water before high-performance liquid chromatographic (HPLC) analysis. Detection is 

performed by means of a tandem mass spectrometer (MS/MS). The deuterated analog of 

cyclophosphamide (d6-CP) is used as an internal standard.

Method Evaluation

This method was evaluated in the general areas of a typical method validation [4, 5]. Furthermore, 

this method has been described by B’Hymer [6] and published by B’Hymer and Cheever [7]. The key 

elements of method validation including accuracy, precision, linearity, specificity, robustness, stability and 

limit of detection have been investigated during this method’s development. The accuracy and precision 

were determined by spiked urine sample recovery studied at four different concentration levels and are 

described in detail later in this report. The other elements are also described in detail with their respective 

results within this report.
Backup Data Report for NMAM 8327: Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine

dated 5 March 2021 - Page 3 of 16

NIOSH Manual of Analytical Methods (NMAM) 5th Edition

Sampling Aspects

All urine samples were prepared by mixing 4.0 mL of urine with 0.5 mL of 2 M potassium 

phosphate buffer solution (pH 7.0). The spiked urine samples were prepared by adding 0.25 mL of the 

appropriate spike-level solution to the buffered urine solution. Non-spiked samples, including the actual 

field samples, were diluted with 0.25 mL of water. All samples were spiked with a 0.25 mL aliquot of 48 

ng/mL deuterated CP, which is the internal standard. Solids in urine samples caused no problems during 

the liquid-liquid extraction; therefore, filtering the urine was not included in this test method’s sample 

preparation procedure.

Analytical Aspects

The chromatographic analysis was carried out using an Agilent Technologies model 1100 HPLC 

pumping system with autosampler (Palo Alto, California, USA) with an Agilent Technologies model 6410A 

triple quadrupole mass spectrometer used as the detector. The detector output was processed by 

Agilent’s Mass Hunter software where all data were evaluated and the chromatographic peaks were 

integrated. The column used was an Agilent Zorbax Rx-C18 (150 X 3 mm, 3.5 μm particle size) using 

gradient elution with acetonitrile/water mobile phases containing 0.1% acetic acid. The chromatographic 

and mass spectrometric conditions were optimized for this method and are described below.

Chromatographic Conditions

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 Program: Time (min) Mobile Phase Comments

 Composition

 0 to 2 0% B Initial hold

 2 to 10 0 to 30% B First linear gradient step

 10 to 18 30 to 100% B Second linear gradient step

 18 to 25 100% B Final hold

 25 to 27 100 to 0% B Start re-equilibration

27 to 35 0% B Column re-equilibration

Injection Volume: 10 μL

Mass Spectrometric Conditions

Ionization Source: Electrospray at 3000 Volts and positive scan mode, nebulizer gas at 35 psi and 

 10 L/min flow

Multiple Reaction Mode (MRM):
Backup Data Report for NMAM 8327: Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine

dated 5 March 2021 - Page 4 of 16

NIOSH Manual of Analytical Methods (NMAM) 5th Edition

Quantification mass transitions - CP = m/z 261 → 140, 4-keto-CP = 275 → 106, IF = 261 → 154, d6-CP = 

267 → 140. 

Qualification mass transitions - CP = m/z 261 → 106, 4-keto-CP = 275 → 204, IF = 261 → 92, 

The following table lists the instrumental settings used for the Agilent 6410A mass spectrometer:

Table 1: Mass spectrometer settings

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

CP 261 wide 106 unit 200 140 25

IF 261 wide 154 unit 200 140 25

IF 261 wide 92 unit 200 140 25

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

4-keto-CP 275 wide 204 unit 200 140 25

Extraction/Sample Preparation

Non-spiked urine samples and those spiked with the three analytes were treated identically. A 4.0 

mL portion of the urine was placed in a screw-capped tube and buffered by the addition of 0.5 mL of 2 M 

potassium phosphate buffer (pH 7). A 0.25 mL aliquot of a 48 ng/mL d6-CP internal standard solution 

was added. A 0.25 mL portion of deionized water or the appropriate analyte spiking solution was added. 

The urine sample was extracted three times with 5.0 mL of ethyl acetate using a vortex mixer for one 

minute for each extraction. The ethyl acetate layers were removed and combined. The extract solution 

was evaporated to dryness by means of a rotary concentrator at 30 °C. Prior to HPLC analysis, the dry 

extract was dissolved in 250 μL of deionized water and transferred to a 100 μL insert in an HPLC 

autosampler vial.

The chemical reagents used were those commonly found in a laboratory. The ethyl acetate was 

HPLC grade. The sources of the reference compounds have been described previously.

Method developmental considerations

Ethyl acetate was chosen for this method due to its improved extraction efficiency for 4-keto-CP

as compared to solid phase extraction and its acceptable extraction efficiency for CP and IF. Recovery 

was found to be 84% (n=6) for 4-keto-CP, 85% for CP and 86% for IF using the method as described 

above. Other extraction procedures were evaluated during the early development stage of this method. 

Various solid-phase extraction procedures were evaluated and included Biotage Isolute ENV+, 
Backup Data Report for NMAM 8327: Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine

dated 5 March 2021 - Page 5 of 16

NIOSH Manual of Analytical Methods (NMAM) 5th Edition

Phenomenex Strata C18 and Varian Bond Elut C18 SPE cartridges. All performed nearly as well for the 

extraction efficiency of CP and IF, but the extraction efficiency was lower for 4-keto-CP (67-69%). Also, it 

was necessary to buffer urine samples to roughly neutral pH to obtain the best recovery of the target 

analytes. Acidic conditions, which is usual for urine, gave lower extraction of CP and IF. The addition of 

0.5 mL of 2 M potassium phosphate buffer (pH 7) to 4 mL of urine was found to give the high extraction 

efficiencies reported.

Metabolites of IF were also evaluated for incorporation into this procedure. It was hoped that 4-

ketoifosfamide (4-keto-IF) could also be studied for possible use as a biomarker for IF exposure. 

Unfortunately, 4-keto-IF was found not to be stable under the aqueous buffer conditions of this procedure 

(pH 7) during this study; it was not stable in human urine under physiological conditions, and therefore, 

was a poor metabolic candidate for use as a biomarker of exposure. 2,3-Dechloroethylifosfamide was 

also initially evaluated during the early stages of method development. 2,3-Dechloroethylifosfamide 

extracted well with SPE, but poorly with various LLE conditions, and its chromatographic recovery was 

poor owing to the lack of an internal standard specific for that compound to compensate for ion 

suppression.

The only other minor difficulty found during the development of this method was with minor 

sample carry-over by the autosampler. The use of a needle rinse with 50/50 (v/v) acetonitrile/water was 

found to be necessary to eliminate this problem.

RESULTS

Accuracy and Precision

Two recovery studies using two columns over several days demonstrated the accuracy and 

precision of this test method. The recovery studies were performed using spiked urine samples containing 

known levels of CP, 4-keto-CP, and IF. The first recovery study was performed over three separate 

experimental batch runs, and these data are presented in Table 2. For each analytical run, the 

experimental trial consisted of three urine samples prepared at three concentration levels. Average 

recovery ranged from 97 to 105% for the three levels of 4-keto-CP investigated, from 101 to 105% for the 

three levels of CP, and from 100 to 102% for the three levels of IF. The recovery results for all three 

analytes are within the statistical expectation and do not appear to have any significant bias. The greatest 

percent relative standard deviation (%RSD) was 7.9% for the 1 ng/mL level of IF. Most of the variation in 

recovery can be attributed to the extraction step within the sample preparation process of this test 

procedure. The secondary recovery experiment used urine samples from 20 non-exposed volunteers, 

and it again demonstrated the procedure to be both accurate and precise (Table 3). The 50 ng/mL 

fortified 4-keto-CP samples had a recovery of 105%. The 2 ng/mL CP and IF fortified samples showed 

recoveries of 104 and 103%, respectively. %RSDs were 8.4% for 4-keto-CP, 2.6% for CP and 7.2% for IF 

for the second recovery experiment. Both recovery experiments generally displayed less precision for 4-

keto-CP and IF, probably owing to the use of only the one deuterated analog of CP as the internal 

standard. The %RSD of 4-keto-CP and IF were within an acceptable range for use as a biomarker assay.
Backup Data Report for NMAM 8327: Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine

dated 5 March 2021 - Page 6 of 16

NIOSH Manual of Analytical Methods (NMAM) 5th Edition

Table 2

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

Analyte/Nominal conc.

 (ng/mL)

Mean measured 

 conc. (n=9)

 (ng/mL)

Average recovery

 (%)

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 chromatographed on three 

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

Zorbax Rx-C18 column was used for experimental batch trials 1 and 2; a second Zorbax Rx-C18 column 

was used on trial run 3.

A %RSD: percent relative standard deviation

Table 3
Backup Data Report for NMAM 8327: Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine

dated 5 March 2021 - Page 7 of 16

NIOSH Manual of Analytical Methods (NMAM) 5th Edition

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

 Analyte mean 

 measured 

 conc. (ng/mL)

Average 

recovery

(%)

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: 4-Keto-CP prepared theoretical concentration was 51.0 ng/mL, CP theory was 2.00 ng/mL and IF 

theory was 2.08 ng/mL

A %RSD: percent relative standard deviation

Linearity

All calibration curves used during the development of this method were found to be linear and had 

coefficients of determination of 0.99 and greater with y-intercepts close to zero. The standard

concentration ranges described were 10 to 625 ng/mL 4-keto-CP equivalent levels and 0.5 to 25 ng/mL 

CP and IF equivalent levels in urine.

Specificity

The optimized chromatographic conditions developed for this procedure proved to be specific,

have no major interferences and enabled simultaneous quantification of the three target analytes. All nonfortified urine samples chromatographed showed no interfering peaks; the blank samples from 20 nonexposed volunteers showed no interferences for CP, IF, 4-keto-CP or the d6-CP internal standard at the 

selected mass transitions used for quantification. The chromatographic baselines displayed little drift from 

the gradient run with the exception of the CP signal, but smaller background peaks did not interfere with 

the three analytes of interest. In the case of m/z 261→ 140 for CP, the baseline drift and noise was less 

significant after approximately 10 minutes into the gradient program. Since CP eluted at approximately 14 

minutes, a reasonable baseline was obtained during its retention time window. Specificity and lack of 

interferences would be expected because of the use of the triple quadrupole mass spectrometer for 

detection.
Backup Data Report for NMAM 8327: Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine

dated 5 March 2021 - Page 8 of 16

NIOSH Manual of Analytical Methods (NMAM) 5th Edition

While qualifying ions were not included in the final method, they were investigated during the 

development of the method. The qualifying mass transition for CP and IF were selected as the second 

highest response that did not have significant interferences with the existing components typically found 

in urine. The qualifying mass transition for 4-keto-CP was chosen for its unique signal for the compound. 

The mass transitions for qualification of the analytes can only be used at higher concentration levels, 

since they have a lower response than the quantifying mass transition ions.

Robustness

Two Agilent Zorbax Rx-C18 columns 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 Zorbax Rx-C18 HPLC column. Recovery results from individual urine samples spiked with the 

three analytes indicate that the method was accurate and not significantly affected by individual urine 

sample matrix differences during analyte extraction or chromatographic analysis.

Stability

Sample stability was not exhaustively evaluated. Stock standard solutions of CP, 4-keto-CP and IF 

were made in acetonitrile and appeared to be stable after weeks of storage under refrigeration. A five-day 

stability study was conducted on the final chromatographic sample solution. The three analytes were

stable at 7 °C (the autosampler temperature) and at room temperature in typical laboratory light (Table 4).

Experiments conducted by Kasel, et al. [8] showed that CP and 4-keto-CP in urine were stable following 

three freeze-and-thaw cycles, were stable at room temperature for the time it takes to process the 

samples (six hours), and that the processed samples were stable after storage in the autosampler tray at 

5 °C for 18 hours. It is reasonable to assume that ifosfamide would behave similarly. Other data also 

show the stability of CP and IF in urine [9,10].

Table 4

Stability of the analytes stored five days at two temperatures

Analyte Recovery stored at 7 °C 

(%)

Recovery stored at room temperature (%)

CP 99.0 97.0

IF 99.1 99.5

4-keto-CP 104.8 100.1

Range 

The procedure was found to be linear within the standard concentration ranges described; 10 to 

625 ng/mL 4-keto-CP equivalent levels and 0.5 to 25 ng/mL CP and IF equivalent levels in urine. Field 

samples at higher levels can be diluted to a concentration within that range for analysis.
Backup Data Report for NMAM 8327: Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine

dated 5 March 2021 - Page 9 of 16

NIOSH Manual of Analytical Methods (NMAM) 5th Edition

Limit of detection

The limit of detection (LOD) was calculated in a traditional manner [1,2] using three times the 

noise level divided by the slope of the calibration curve. Since instrumental noise is a function of height, 

the average baseline level of height noise was determined for each batch run in chromatograms at the 

retention time window for each analyte from the blank samples. This was done by exporting raw data files 

into Microsoft Excel® and determining the mean height level and the standard deviation of height noise 

from 100 data points within the retention time window noted for specific analyte monitoring the specific 

transition signal. The slope from the calibration curve using peak heights of all the standard solutions was 

determined and then used as the divisor for this LOD calculation. It should be noted that peak height was 

used only for the estimation of the LOD; the peak area ratio was used for quantification of the analytes 

during the validation of this procedure. This “instrumental” LOD was found to be approximately 1 ng/mL 

for 4-keto-CP, 0.1 ng/mL for CP and 0.05 ng/mL for IF. The actual LOD is dependent upon the

performance of the chromatographic system and the detector at the time of the analysis and the 

extraction efficiency. Since this method calls for the lowest standard concentrations of 0.5 ng/mL for CP 

and IF and 10 ng/mL for 4-keto-CP, these can be considered the “operational” LODs and a basic criteria 

for the use of this method. If a column, chromatographic system or detector cannot detect the lowest 

standard level, corrective action would be required.

Ruggedness

Laboratory-to-laboratory reproducibility was evaluated through the User Check process. A 

laboratory was contracted to set up the method in their laboratory, show analytical proficiency, and then 

analyze a set of blind spiked samples. The results of this User Check and discussion of it are found in the 

Appendix.

References

1. Li F, Patterson AD, Hofer CC, Krausz KW, Gonzalez FJ, Idle JR [2010]. Comparative metabolism of 

cyclophosphamide and ifosfamide in the mouse using UPLC-ESI-QTOFMS-based metabolomics. 

Biochem Pharmacol 80:1063-1074.

2. DrugBank [2018]. Cyclophosphamide. www.drugbank.ca/drugs/DB00531.

3. Joqueviel C, Gilard V, Martino R, Malet-Martino M, Niemeuyer U [1997]. Urinary stability of 

carboxycyclophosphamide and carboxyifosfamide, two major metabolites of the anticancer drugs 

cyclophosphamide and ifosfamide. Cancer Chemother Pharmacol 40:391-399.

4. Green JM [1996]. A practical guide to analytical method validation. Anal Chem 68:A305-A309.

5. FDA [2018]. Guidance for industry. Bioanalytical method validation. U.S. Food and Drug 

Administration, Rockville, MD. http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf.

6. B’Hymer C [2008]. Determination of cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in 

urine. NIOSH, Division of Applied Research and Technology, Biomonitoring and Health Assessment 
Backup Data Report for NMAM 8327: Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine

dated 5 March 2021 - Page 10 of 16

NIOSH Manual of Analytical Methods (NMAM) 5th Edition

Branch SOP no. E117.

7. 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.

8. Kasel D, Jetter A, Harlfinger S, Gebhardt W, Fuhr U [2004]. Quantification of cyclophosphamide and 

its metabolites in urine using liquid chromatography/tandem mass spectrometry. Rapid Commun Mass 

Spectrom 18:1472-1478.

9. Sottani C, Tranfo G, Faranda P, Minoia C [2005]. Highly sensitive high-performance liquid 

chromatography/selective reaction monitoring mass spectrometry method for the determination of 

cyclophosphamide and ifosfamide in urine of health care workers exposed to antineoplastic agents. Rapid 

Commun Mass Spectrom 19:2794-2800.

10. Hartley JM, Hansen L, Harland SJ, Nicholson PW, Pasini F, Souhami RL [1994]. Metabolism of 

ifosfamide during a 3 day infusion. Br J Cancer 69:931-936.
Backup Data Report for NMAM 8327: Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine

dated 5 March 2021 - Page 11 of 16

NIOSH Manual of Analytical Methods (NMAM) 5th Edition

Appendix

Review of User Check for NMAM Method 8327 (Cyclophosphamide, 4-ketocyclophosphamide, and 

ifosfamide in urine)

User check samples were prepared by a BHAB researcher to be analyzed by a contract lab using draft 

NMAM Method 8327. A total of 25 urine samples were prepared. The urine was obtained from personnel 

in the Taft building at NIOSH and then combined and mixed in the BHAB labs into a single pool of urine 

from which all samples were prepared. Five samples were left blank. Five samples were prepared 

containing the analytes at each of the following levels: cyclophosphamide (CP) – 0.988 ng/mL, 1.98 

ng/mL, 9.88 ng/mL, and 19.8 ng/mL; 4-ketocyclophosphamide (4-ketoCP) – 26.1 ng/mL, 52.2 ng/mL, 261 

ng/mL, and 522 ng/mL; ifosfamide (IF) – 1.02 ng/mL, 2.04 ng/mL, 10.2 ng/mL, and 20.4 ng/mL. Thus the 

lowest level sample contained 0.988 ng/mL CP, 26.1 ng/mL 4-ketoCP, and 1.02 ng/mL IF and so forth for 

the other three concentration levels. The samples were prepared, shipped frozen on July 26, 2017, and 

arrived at the contract lab the next day. The samples were analyzed on August 7, 2017. The results are

in Tables 1a-3a.

For this analysis, the Lower Limit of Quantitation (LLOQ) was determined by the lab to be ~0.5 ng/mL for 

CP and IF and ~10 ng/mL for 4-ketoCP. As mentioned above, the spike levels ranged from 1 to 20 ng/mL 

for CP and IF and from 25 to 500 ng/mL for 4-ketoCP which are 2 to 40 times the LLOQ and fall within the 

method detection range for all three analytes. The urine blanks showed no levels of any of the 

compounds, thus no adjustment was needed.

Summary tables for each analyte are shown below. Larger tables containing each individual sample value 

can be found at the end of this report as Tables 4a-6a.

Table 1a. Summary Cyclophosphamide user check results

Spiked 

amount 

(ng/mL)

Average 

recovery 

(%)

RSD 

(%)

19.8 111 2.6

9.88 114 1.9

1.98 120 2.8

0.988 120 3.3

Overall 116 4.3
Backup Data Report for NMAM 8327: Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine

dated 5 March 2021 - Page 12 of 16

NIOSH Manual of Analytical Methods (NMAM) 5th Edition

Table 2a. Summary 4-Ketocyclophosphamide user check results

Spiked 

amount 

(ng/mL)

Average 

recovery 

(%)

RSD 

(%)

522 100 4.1

261 111 3.5

52.2 119 1.9

26.1 114 4.3

Overall 111 7.1

Table 3a. Summary Ifosfamide user check results

Spiked 

amount 

(ng/mL)

Average 

recovery 

(%)

RSD 

(%)

20.4 102 3.3

10.2 104 1.7

2.04 111 3.9

1.02 112 4.2

Overall 107 5.4

Two of the primary guidelines on bioanalytical method validation recommend that accuracy and precision 

should be within ± 15% at each level and within ± 20% at the lowest level [1,2]. The precision, as 

measured by relative standard deviation, fall within this range for every level of every analyte as well as 

for the overall precision across every sample for all three analytes. As a further acceptability proof of the 

precision of the method, the contract lab found that the inter-run RSD for the urine standards were 10% or 

less at all concentrations, that the intra-run RSDs for the urine quality control (QC) samples were 10% or 

less for all three compounds, and that the inter-run RSDs for the urine QC samples were within 10% for 

all three compounds. These inter-run values are produced over three separate analysis batches with four 

replicates of QC samples at three concentrations and performed by two different analysts.

The recovery values (accuracy) are slightly more mixed. The same three separate analysis batches 

mentioned above produced the following data. The intra-run relative errors (REs) for the urine standards 

were 10 percent or less at all concentrations for all compounds except the LLOQ which was up to -11.5 

percent for IF and -10.6 percent for 4-keto-CP. The inter-run REs for the urine standards for all runs were 

10 percent or less for all concentrations for all compounds. The intra-run REs for the urine QCs for all 

runs were 12 percent or less for all concentrations for CP and IF. The intra-run REs for the urine QCs for 

all 4-Keto-CP runs were 15.8 percent or less for all concentrations. The inter-run REs for the urine QCs 

were within 10 percent for all compounds.

For the blind User Check samples, the average recovery for ifosfamide is also within the desired range at 

every level and for the average of every sample, with a range of recovery from 96% to 120%. The 
Backup Data Report for NMAM 8327: Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine

dated 5 March 2021 - Page 13 of 16

NIOSH Manual of Analytical Methods (NMAM) 5th Edition

accuracy values for CP are slightly outside the recommended range at the next to lowest level and right 

at the top of the range at the lowest level with recoveries ranging from 106% to 125%. The values for 4-

keto-CP are also slightly above the recommended range at the next to lowest level with recoveries 

ranging from 97% to 122%. While the high recoveries at a couple of individual levels are somewhat less 

than desirable for this User Check, the multitude of the data in this User Check, in the method 

development, and in the contract lab’s multi-day runs demonstrate an acceptable level of precision and 

accuracy.

The contract lab reported no difficulties understanding the draft method nor in setting it up or analyzing 

the samples. The method has relatively few analytical steps, is quite straightforward, is sensitive enough 

to determine occupational exposures to CP and IF, and has been shown to have adequate precision and 

accuracy. It is recommended that the method, NMAM Method 8327 (Cyclophosphamide, 4-

ketocyclophosphamide, and ifosfamide in urine) be approved and accepted for inclusion in the NIOSH 

Manual of Analytical Methods.

Dale Shoemaker, PhD

Research Chemist

August 10, 2018

References

[1] European Medicines Agency [2011]. Guideline on bioanalytical method validation. 

http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC50010

9686.pdf.

[2] FDA [2018]. Guidance for industry. Bioanalytical method validation. U.S. Food and Drug 

Administration, Rockville, MD. http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf.
Backup Data Report for NMAM 8327: Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine

dated 5 March 2021 - Page 14 of 16

NIOSH Manual of Analytical Methods (NMAM) 5th Edition

Table 4a. Cyclophosphamide user check results

CP 

found 

(ng/mL)

Target 

(ng/mL)

Recovery 

(%)

22.6 19.8 114.1

22.1 19.8 111.6

21.9 19.8 110.6

22.2 19.8 112.1

20.9 19.8 105.6

10.9 9.88 110.3

11.5 9.88 116.4

11.3 9.88 114.4

11.5 9.88 116.4

11.3 9.88 114.4

2.38 1.98 120.2

2.43 1.98 122.7

2.44 1.98 123.2

2.34 1.98 118.2

2.26 1.98 114.1

1.19 0.988 120.4

1.24 0.988 125.5

1.20 0.988 121.5

1.19 0.988 120.4

1.12 0.988 113.4
Backup Data Report for NMAM 8327: Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine

dated 5 March 2021 - Page 15 of 16

NIOSH Manual of Analytical Methods (NMAM) 5th Edition

Table 5a. 4-Ketocyclophosphamide user check results

4-

ketoCP 

found 

(ng/mL)

Target 

(ng/mL)

Recovery 

(%)

565 522 108.2

516 522 98.9

517 522 99.0

516 522 98.9

504 522 96.6

281 261 107.7

303 261 116.1

281 261 107.7

279 261 106.9

298 261 114.2

62.8 52.2 120.3

62.2 52.2 119.2

63.2 52.2 121.1

59.8 52.2 114.6

62.3 52.2 119.3

29.0 26.1 111.1

30.5 26.1 116.9

28.2 26.1 108.0

29.6 26.1 113.4

31.9 26.1 122.2
Backup Data Report for NMAM 8327: Cyclophosphamide, 4-ketocyclophosphamide and ifosfamide in urine

dated 5 March 2021 - Page 16 of 16

NIOSH Manual of Analytical Methods (NMAM) 5th Edition

Table 6a. Ifosfamide user check results

IF found 

(ng/mL)

Target 

(ng/mL)

Recovery 

(%)

21.5 20.4 105.4

21.3 20.4 104.4

20.9 20.4 102.5

20.4 20.4 100.0

19.6 20.4 96.1

10.4 10.2 102.0

10.8 10.2 105.9

10.4 10.2 102.0

10.7 10.2 104.9

10.8 10.2 105.9

2.24 2.04 109.8

2.36 2.04 115.7

2.38 2.04 116.7

2.19 2.04 107.4

2.16 2.04 105.9

1.08 1.02 105.9

1.22 1.02 119.6

1.15 1.02 112.7

1.16 1.02 113.7

1.11 1.02 108.8