NIOSH Manual of Analytical Methods (NMAM) 5th Edition 

BACKUP DATA REPORT

NIOSH Method No. 9212

Title: Mitomycin C on Surfaces

Analyte: Mitomycin C

Author/developer: Clayton B’Hymer and Thomas H. Connor

Date: 10/16/2014

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. 
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NIOSH Manual of Analytical Methods (NMAM) 5th Edition

BACKUP DATA REPORT

NIOSH Method 9212

Mitomycin C on surfaces

Clayton B’Hymer and Thomas H. Connor

October 16, 2014

Substance: Mitomycin C

 Exposure Limits: Not applicable

Chemicals Used for Evaluation:

1. Mitomycin C ([6-Amino-8a-methoxy-5-methyl-4,7-dioxo-1,1a,2,4,7,8,8a,8b-octahydroazireno

 [2',3':3,4]pyrrolo[1,2-a]indol-8-yl]methyl carbamate, CAS no. 50-07-7) from the United States 

 Pharmacopeia (Rockville, Maryland, USA), 98%, Lot no. K.

2. Porfiromycin ([1aS-(1aα,8β,8aα,8bα)]-6-Amino-8-[[(aminocarbonyl)oxy]methyl]-1,1a,2,8,8a,8b-

 hexahydro-8a-methoxy-1,5-dimethylazirino[2′,3′:3,4]pyrrolo[1,2-a]indole-4,7-dione, CAS no. 801-52-5) 

 from the National Cancer Institute, Lot no. 56410/3.

General

Synopsis

Mitomycin C is an alkylating agent and an antibiotic used as a chemotherapeutic agent because of 

its antitumor properties. Mitomycins are part of a family of aziridine containing natural products first 

isolated from the soil bacteria Streptomyces caespitosus and Streptomyces lavendulae. Mitomycin C is

used occasionally in the intravenous treatment mode and used extensively in the topical treatment mode 

of cancer. It has also been used in instillation theory in the treatment of bladder tumors and has come into 

use of intraperitoneal chemotherapy procedures including Hyperthermic Intraperitoneal Chemotherapy 

(HIPEC) and Continuous Hyperthermic Peritoneal Perfusion (CHPP) therapy. Both of the latter 

chemotherapeutic procedures involve possible exposure to surgical staff by contamination of the 

operating room. The described procedure was developed for the detection and quantification of 

mitomycin C from contaminated surfaces typically encountered in an operating room. In summary, a 100 

cm2 template surface area is sampled by wetting the surface and then wiping the surface with either a 

Texwipe® swab or filter paper three times. The sampling media is placed in a polypropylene jar, extraction 

solvent and the porfiromycin internal standard are added and the jar swirled for 30 minutes. The extract 

solution is filtered and high-performance liquid chromatographic analysis is performed. Detection is by 

means of a tandem mass spectrometer (MS/MS).

Method Evaluation

This method was evaluated in the general areas recommended for typical method validation [1,2]. 

Furthermore, this method has been described by B’Hymer and Connor [3] and B’Hymer, et al. [4]. 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 matrix matched solutions and spiked surfaces. Recoveries were

studied at up to six different concentration levels on two different HPLC-MS/MS systems which are 

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

results within this report.
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Sampling Aspects and Sample Preparation

Potential mitomycin C contamination was evaluated for three work surfaces using two separate 

wiping materials. The first wipe procedure evaluated for collecting and processing samples utilized a 

simple paper filter disk procedure used previously by NIOSH [5]. A 0.25 mL aliquot of the extraction 

solvent (20/45/35 (v/v/v) acetonitrile-isopropanol-water made 0.01 M in ammonium citrate, apparent pH 

7.0) was used to wet a template area of 100 cm2 on the desired surface. A filter paper disc (55 mm 

Whatman 42, GE Healthcare, Buckinghamshire, UK) was used to wipe the template area. This wiping 

procedure was conducted three times over the surface area. The three filters were then placed in a 125 

mL polypropylene jar (Nalgene/Thermo Scientific Co., part number 2118-004, Rochester, NY, USA) and 

brought up to a total liquid volume of 9 mL with the extraction solvent. A 1.0 mL aliquot of the internal 

standard solution was added to the jar and it was swirled by means of an orbital shaker for 30 minutes to 

extract the mitomycin C. The approximate 10 mL volume of solvent was an optimum to cover the three 

pieces of filter paper in each jar. The second wipe procedure evaluated used polyester Texwipe® swabs 

(TX714A, Large Alpha Swab, ITW Texwipe, Kernersville, NC, USA). Similar to the filter paper, 0.25 mL 

aliquots of the extraction solvent were used and three swabs were used to wipe the template area. After 

wiping, the heads were removed from the swab shafts and placed in the 125-mL polypropylene jars. The

swabs were treated by the same procedure as the filter paper. After the analytes were extracted into the 

solvent, the extracts were filtered using Millex 0.22 μm polyvinylidene difluoride filters (Millipore 

Corporation, Billerica, MA, USA) and placed in autosampler vials for analysis.

The recoveries for the full surface wiping procedure were evaluated by selecting three materials to 

represent surfaces likely to experience contamination in the workplace. The three materials selected were 

stainless steel, vinyl and Formica®. Stainless steel (Type 304 #3 satin finish, McMaster-Carr Supply, 

Cleveland, OH, USA) was used as a surrogate for biological safety cabinets, Formica® for counter tops 

and vinyl for floor tiles (Imperial Texture, Armstrong World Industries, Montreal, Quebec, Canada). Tiles 

were cut into 10 X 10 cm sections from sheets of stock materials, washed with methanol and air dried 

prior to use. Recovery studies from the surfaces were conducted by spiking the surfaces with known 

amounts of mitomycin C dissolved in 250 μL of acetonitrile. The acetonitrile was allowed to evaporate 

before wiping the surface for the recovery study. For all the recovery studies, both the spiked solutions 

and the spiked surfaces, equivalent mitomycin C levels of 0.1, 0.2, 2, 4, 10, 25 μg/100 cm2 were 

prepared. Sample solutions prepared outside the range of the calibration curves [the 10 and 25 μg/100 

cm2 samples] were diluted 1 to 10 in extraction solvent before analysis.

Analytical Aspects

The chromatographic analysis was carried out using two liquid chromatography-tandem mass 

spectrometry systems. One was an Agilent Technologies model 1100 HPLC pumping system with 

autosampler (Palo Alto, California, USA) with an Agilent Technologies model 6430 triple quadrupole mass 

spectrometer used as the detector. The detector output and data were evaluated by Agilent’s Mass 

Hunter software. The second system consisted of a Waters Corporation Aquity HPLC pumping system 

with autosampler (Milford, Massachusetts, USA) with a Waters Xevo TQ mass spectrometer used as the 

detector. The Waters system utilized their MassLynx software where all data were evaluated and the 

chromatographic peaks integrated. The type of analytical column used in this method was an Agilent 

Zorbax RX C18 (150 X 3mm, 3.5 μm particle size) along with a guard column consisting of a 

Phenomenex C18 SecurityGuard (4 X 2 mm). An SSI 2 micron precolumn filter was also found to be

necessary for this procedure. Gradient elution with acetonitrile/water mobile phases both at 0.1% acetic 

acid was used. An example chromatogram is displayed in Figure 1. The chromatographic and mass 

spectrometric conditions were optimized for this method on each instrument and are described next.
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NIOSH Manual of Analytical Methods (NMAM) 5th Edition

Chromatographic Conditions

Mobile Phases: A = 10/90/0.1% (v/v/v) acetonitrile/water/acetic acid

 B = 75/25/0.1% (v/v/v) acetonitrile/water/acetic acid

Flow Rate: 0.4 mL/min (during analysis time)

Gradient Program: Time (minutes) Mobile Phase Comments

 Composition

0 to 12 0 to 70% B Gradient ramp

12 to 13 70 to 100% B Start post run 

13 to 16 100% B Post run [Increase flow to 0.5 mL/min]

16 to 18 0 to 100% A Decrease flow to 0.4 mL/min 

18 to 24 100% A Column re-equilibration

Injection Volume: 5 μL

Autosampler temperature held at 8 °C

Agilent Model 6430 Mass Spectrometric Conditions:

Ionization Source: Electrospray at 3500 Volts and positive scan mode, the nitrogen nebulizer gas at 

 35 psi 

Drying Gas Flow: 10 L/min

Drying Gas temperature: 325°C

Multiple Reaction Mode (MRM)

Collision gas: Nitrogen

Quantification mass transitions: Mitomycin C = m/z 335 → 242, Porfiromycin (int. std.) = m/z 349 → 256

The following table lists the instrumental settings used for the Agilent 6430 mass spectrometer:

Analyte Precursor 

Ion

(m/z)

MS1

Resolution

Product

Ion

(m/z)

MS2

Resolution

Dwell

Time 

(msec)

Fragmentor

Voltage

Collision

Energy

Cell

Accelerator

Voltage

Mitomycin C 335 unit 242 unit 200 97 8 4

Porfiromycin 349 unit 256 unit 200 97 6 4

Waters Corporation Xevo TQ Mass Spectrometric Conditions:

Ionization Source: Electrospray at 3.5 Kilovolts and positive scan mode

Desolvation gas: 650 L/hr

Drying Gas temperature: 350 °C

Multiple Reaction Mode (MRM)

Collision gas: Argon

Quantification mass transitions: Mitomycin C = m/z 335 → 242, Porfiromycin (int. std.) = m/z 349 → 256, 

mass span set at 1.0
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The following table lists the instrumental settings used for the Waters Xevo TQ mass spectrometer:

Analyte Parent ion 

(m/z)

Daughter ion 

(m/z)

Dwell (s) Cone (V) Collison 

Energy (V)

Mitomycin C 335 242 0.100 17 15

Porfiromycin 349 256 0.100 17 16

Another important aspect of this method was the use of a 2 micron precolumn filter. It was found 

that a small amount of additional mobile phase dead volume was necessary to maintain good peak shape 

owing to the use of a high organic content and mismatch with the initial mobile phase conditions. The 

sample wiping solution's high organic content was necessary owing to the stability of target mitomycin C 

analyte. The stability problems will be addressed in the next section of this report.

RESULTS

Accuracy and Precision

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

precision of this test method. These matrix matched samples were made to mimic matrices that would be 

found in the field. Blank surfaces were wetted and the surfaces sampled as in Steps 3 or 4 of the 

Sampling Section of the method. The solutions containing the three wipes (filter paper or swab heads) 

were then spiked with varying amounts of the analyte and carried through the analytical process. A 

primary recovery experiment using the optimized conditions with filter paper matrix matched solution

fortified with mitomycin C was performed to demonstrate the general accuracy and precision of the HPLC 

conditions using the Agilent mass spectrometer. These data are presented in Table 1. Average 

recoveries ranged from 93 to 105% for the six fortified levels of mitomycin C investigated. A similar 

recovery study using the Texwipe® swab matrix matched solution fortified with mitomycin C was 

performed using the Waters Xevo mass spectrometer. These data are presented in Table 2. Average 

recoveries ranged from 90 to 107% for the four fortified levels of mitomycin C investigated. These results 

are well within the acceptable guidelines for the accuracy and precision of an analytical test method [2].
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Table 1

Recovery of Mitomycin C Spikes Added to Filter Paper Matrix-Matched Extracts

(Analysis was performed using an Agilent Model 6430 Mass Spectrometer)

Surface Equivalent Level

(μg/100 cm2)

Mean

(n=10)

% Recovery % RSD

(n=10)

Stainless Steel 0.100 0.104 104 7.5

 0.200 0.202 100 3.3

 2.00 2.01 100 1.0

 4.00 4.08 102 2.7

 10.0 9.81 98 2.5

 25.0 23.2 93 5.1

Vinyl 0.100 0.105 105 3.0

 0.200 0.201 101 2.3

 2.00 1.99 100 3.1

 4.00 4.06 102 2.6

 10.0 9.82 98 2.7

 25.0 23.8 95 5.9

Formica® 0.100 0.103 103 3.3

 0.200 0.200 100 2.7

 2.00 2.02 101 3.0

 4.00 4.05 101 1.8

 10.0 9.66 97 2.0

 25.0 23.6 94 5.4

Notes: This recovery study was performed using two different Zorbax columns. RSD is relative standard 

deviation.

Table 2

Recovery of Mitomycin C Spikes Added to Texwipe® Swab Matrix-Matched Extracts

(Analysis was performed using a Waters Xevo TQ Mass Spectrometer)

Surface Equivalent Level

(μg/100 cm2)

Mean

(n=5)

% Recovery % RSD

(n=5)

Stainless Steel 0.100 0.090 90 1.3

 0.200 0.187 94 1.4

 2.00 2.02 101 4.1

 4.00 4.20 105 2.1

Vinyl 0.100 0.105 105 2.4

 0.200 0.200 100 1.3

 2.00 1.99 99 1.0

 4.00 3.96 99 1.1

Formica® 0.100 0.107 107 1.7

 0.200 0.214 107 0.8

 2.00 2.06 103 1.2

 4.00 4.21 105 0.8

The most important aspect of this method was to evaluate the most effective wipe sampling 

media to detect mitomycin C from various surfaces encountered in the operating room setting. The data 

for the surfaces tested after spiking the actual surface and wiping using either filter paper or the polyester 
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NIOSH Manual of Analytical Methods (NMAM) 5th Edition

swabs are presented in Table 3. As can be seen in the table, stainless steel generally has higher 

recovery yields with some less than quantitative recoveries at the lower levels. There was little difference 

in using filter paper or swabs for extracting mitomycin C from stainless steel. Vinyl is a porous material 

and it generally had recovery yields of 50 to 60% for every spike level using either filter paper or swabs 

for the wipe procedure. Finally, the main quantitative advantage for using the swab over filter paper can 

be seen for the Formica® surface data (see Table 3). The swabs had recovery yields of 60 to 70% at the 

0.100 and 0.200 μg/100 cm2 levels while the filter paper had recoveries near 30%. The swabs also 

displayed greater precision at the lower mitomycin levels (see Table 3), although the recoveries were still 

less than quantitative at the two lower concentration levels studied.
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Table 3

Recovery from Spiked Material Surfaces

Surface Filter Paper Wipe Texwipe® Swab

Level

(μg/100 cm2)

Mean

(n=5)

% Recovery % RSD Level

(μg/100 cm2)

Mean

(n=5)

% Recovery % RSD

Stainless Steel 0.100 0.068 68 6.3 0.100 0.071 71 6.4

 0.200 0.124 62 10.8 0.200 0.122 61 5.6

 2.00 1.75 88 3.6 2.00 1.77 89 3.6

 4.00 3.67 92 3.8 4.00 3.76 94 2.5

 10.0 9.80 98 8.3 10.0 9.76 98 1.6

 25.0 23.9 96 3.1 25.0 24.8 99 2.6

Vinyl 0.100 0.058 58 7.9 0.100 0.062 62 6.2

 0.200 0.115 58 12.6 0.200 0.109 54 3.7

 2.00 1.12 56 7.9 2.00 1.32 66 4.3

 4.00 2.02 51 8.0 4.00 2.39 60 5.9

 10.0 6.25 63 3.4 10.0 5.32 53 8.2

 25.0 14.8 59 7.6 25.0 14.0 56 5.1

Formica® 0.100 0.030 30 47 0.100 0.073 73 7.9

 0.200 0.065 33 6.0 0.200 0.126 63 5.3

 2.00 1.53 77 8.5 2.00 1.74 87 1.2

 4.00 3.27 82 1.2 4.00 3.65 91 1.7

 10.0 9.05 91 2.0 10.0 9.26 93 2.9

 25.0 24.0 96 3.7 25.0 24.3 97 0.7

NOTE: Analysis was performed using an Agilent Model 6430 Mass Spectrometer.
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Linearity, Range and LOD

The linearity of this methodology was determined over the range of 0.002 to 0.5 μg/mL (0.02 to 5 

μg/100 cm2 spiked equivalent level). The calibration curves generated from the peak area ratio between 

mitomycin C and the internal standard showed correlation of determinations (r2) of 0.99 or greater. As to 

the limit of detection (LOD), it should be noted that the actual LOD is dependent upon the performance of 

the chromatographic system and the detector at the time of an analysis. The lowest standard used was 

equivalent to 20 ng/100 cm2 in the calibration curves and can be used as the LOD for wipe testing. If a 

column, chromatographic system or detector cannot detect the lowest standard level, corrective action 

would be required. During the development stage of this method, the instrumental LOD was determined 

in the traditional manner; three times the noise level of the chromatogram’s baseline divided by the slope 

of a calibration curve using peak heights. This level was found to be approximately 2 ng/100 cm2

equivalent (0.002 μg/100 cm2) for the Agilent mass spectrometer. The Waters mass spectrometer was 

determined to have a lower LOD. Since the lowest standard is typically the one used as an operational 

LOD, both instruments were readily capable of meeting that level.

Specificity

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

have no interferences and enabled the accurate quantification of the target analyte. All non-fortified blank 

matrix samples chromatographed showed no interfering peaks for either mitomycin C or the internal 

standard. The chromatographic baselines displayed little drift during the gradient run and proved to be 

easily integrated for data analysis. An example chromatogram is displayed in Figure 1.

Figure 1

Chromatogram of a sample taken from a spiked matrix solution. The mitomycin C and 

porfiromycin solution concentrations are approximately 20 ng/mL.

Robustness

Two Agilent Zorbax RX C18 columns were used during the filter paper matrix matched recovery 

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

reproducible with any functioning Zorbax RX HPLC column. Two different mass spectrometric detectors 

both gave reasonable recovery results.
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Stability

Mitomycin C exhibits antibiotic properties in addition to its antineoplastic one; it has been used in 

various pharmaceutical preparations including ophthalmic formulations. As such, its stability has been 

well established in the chemical literature. It can be easily hydrolyzed under acidic or basic conditions, but 

it is generally stable in a pH range between 7 and 8. Also, the use of aprotonic solvents or 

organic/aqueous solvents, such as mixtures of propylene glycol and water, which do not promote acid or 

base catalyzed hydrolysis of mitomycin C have been utilized and reported [6]. The acetonitrileisopropanol-water system developed for the current method was found to be a good matrix for sample 

stability, and it was experimentally determined to be superior to the propylene glycol-water mixtures 

reported in patented pharmaceutical formulations [5]. Mitomycin C stability concerns also limited the 

amount of sample manipulation and preparation possible with this method. Early sample preconcentration experiments including typical simple solvent evaporation demonstrated significant 

degradation of the analyte. Therefore, the method ultimately developed has been limited to simple 

filtration of the extraction solvent before HPLC-MS/MS analysis.

Mitomycin C solutions stored in the acetonitrile-isopropanol-water extraction solvent at room 

temperature in darkness and those held at 8 oC in the autosampler were found to degrade only 3 to 4% 

after 8 days (n=3) (Table 4). The optimized extraction solvent also gave excellent recoveries during the 

surface wipe testing. Stock solutions of mitomycin C stored in 100% acetonitrile were found to only 

degrade 2 to 3% after 3 to 6 months of storage at -15 oC. Mitomycin C did show some light sensitivity. 

Sample solutions (n=3) made in the 20/45/35 (v/v/v) acetonitrile-isopropanol-water 0.01 M ammonium

citrate (pH 7) extraction solvent degraded 12% after 8 days exposed to window sunlight and the 

laboratory fluorescent lights (Table 4). From these experiments, it was deemed advisable to make an 

effort to minimize light exposure of mitomycin C solutions. Amber autosampler vials were used for all 

recovery studies while developing this method.

Table 4

Mitomycin C solution stability

Condition/Time 24 hours 72 hours 8 days

Clear Vials - Light 98.3 95.7 87.9

Darkness 97.5 96.2 95.6

Amber vials at 8oC 98.2 96.6 95.9

Ruggedness

This method was originally developed for support of healthcare worker studies within the 

Biomonitoring and Health Assessment Branch (BHAB), Division of Applied Research and Technology 

(DART) at NIOSH. Reproducibility within the laboratory over the period of method validation was 

performed and these data are shown in Tables 1, 2 and 3. Laboratory-to-laboratory reproducibility of the 

analytical procedure was evaluated through the User Check process. An independent laboratory set up 

the method, spiked wipes and swabs at four different concentration levels, and analyzed them according 

to NIOSH Method 9212. The report of the User Check can be found in the Appendix. Acceptable values 

for precision and accuracy were obtained in the User Check process showing the method to be rugged 

and translatable to other laboratories. 
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NIOSH Manual of Analytical Methods (NMAM) 5th Edition

References

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

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

Administration, Rockville, MD, https://www.fda.gov/media/70858/download.

[3] B’Hymer C, Connor TH [2014]. Determination of mitomycin C contamination from surfaces by HPLCMS/MS. NIOSH, Division of Applied Research and Technology, Biomonitoring and Health Assessment 

Branch SOP no. E3-131.

[4] B’Hymer C, Connor T, Stinson D, Pretty J [2015]. Validation of an HPLC-MS/MS and wipe procedure 

for mitomycin C contamination. J Chromatogr Sci 53:619-624.

[5] Connor TH, DeBord DG, Pretty JR, Oliver MS, Roth TS, Lees PSJ, Krieg EF Jr., Rogers B, Escalante 

CP, Toennis CA, Clark JC, Johnson BC, McDiarmid MA [2010]. Evaluation of antineoplastic drug 

exposure of health care workers at three university-based US cancer centers. J Occup Environ Med 

52:1019-1027.

[6] Velpandian T, Saluja V, Ravi AK, Kumari SS, Mathur R, Biswas NR, Ghose S [2005]. Evaluation of 

the stability of extemporaneously prepared ophthalmic formulation of mitomycin C. J Ocul Pharmacol 

Ther 21:217-222.
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Appendix

Review of User Check for NIOSH Method 9212 (Mitomycin C on surfaces)

User Check samples (swabs and wipes) were prepared by contract lab personnel blind to the analyst. 

The swab samples and the filter paper wipe samples were prepared separately, but at the same 

concentration levels. A total of 25 swab samples and 25 wipe samples were prepared. Five samples of 

each kind were left blank. Five fortified samples of each kind were prepared containing the analyte at 

each of the following levels: 5 ng, 10 ng, 50 ng, and 400 ng. The samples were then analyzed using the 

sample preparation procedures and analytical detection conditions found in draft NIOSH method 9212.

These samples were all spiked directly on to the two-wipe media. No surfaces were analyzed.

Tables 1 (swabs) and 2 (wipes) show the data obtained from the User Check samples. Mitomycin C was 

not detected in any of the blank samples (data not shown), so no corrections were required. Summary 

Tables 3 (swabs) and 4 (wipes) show the average recoveries (at each level and total) and precision as 

calculated by relative standard deviation (RSD).

Table 1 (mitomycin C on swabs)

Spike Level 

(ng) Recovery (ng)

Accuracy 

(%)

5 4.97 99.4

5 4.95 99.0

5 5.06 101.2

5 5.28 105.6

5 4.47 89.4

10 8.92 89.2

10 9.51 95.1

10 10.3 103.0

10 10.1 101.0

10 10.4 104.0

50 49.0 98.0

50 50.3 100.6

50 47.1 94.2

50 48.2 96.4

50 48.9 97.8

400 420 105.0

400 413 103.3

400 436 109.0

400 413 103.3

400 409 102.3
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Table 2 (mitomycin C on filter paper)

Spike Level 

(ng) Recovery (ng)

Accuracy 

(%)

5 5.22 104.4

5 5.26 105.2

5 5.10 102.0

5 5.35 107.0

5 5.02 100.4

10 8.94 89.4

10 9.76 97.6

10 9.63 96.3

10 9.76 97.6

10 9.37 93.7

50 46.4 92.8

50 48.0 96.0

50 48.2 96.4

50 49.7 99.4

50 48.5 97.0

400 413 103.3

400 374 93.5

400 412 103.0

400 427 106.8

400 420 105.0

Table 3 (Average recovery and relative standard deviation - swabs)

Spike level (ng) Recovery (%) RSD (%)

5 98.9 6.0

10 98.5 6.3

50 97.4 2.4

400 105 2.6

Overall 99.8 5.1

Table 4 (Average recovery and relative standard deviation – filter paper)

Spike level (ng) Recovery (%) RSD (%)

5 104 2.5

10 94.9 3.7

50 96.3 2.5

400 102 5.0

Overall 99.3 5.1

The accuracy at every concentration level for both swabs and wipes is within ± 5% of the true value, 

which is well within acceptable levels. The RSD for each individual level ranges between 2 and 7 percent. 

These precision values, as well as the overall precision values, are also well within acceptable limits. Two 

primary guidelines on method validation state that accuracy and precision be within 15% at each level 

and within 20% at the lowest level [1,2].
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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, has been shown to 

give reasonable recoveries for different surfaces, and has adequate analytical precision and accuracy. It 

is recommended that the method, NIOSH 9212 (Mitomycin C on surfaces) be approved and accepted for 

inclusion in the NIOSH Manual of Analytical Methods.

Dale A. Shoemaker, Ph.D.

Research Chemist

April 5, 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, https://www.fda.gov/media/70858/download.