Case Study: Sterilization of Surgical Instruments

Analysis of Cleaning Residues on
Medical Instruments
Case Study
Released by:
Mark Jordi, Ph.D.
President
Job Number: J8906
Page 1 of 35

May 28, 2015
Customer Phone
Contact Email
Address
Dear Valued Customer,
Please find enclosed the test results for your samples described as:
1. Surgical Needle Clamp
The following test was performed:
1. Liquid Chromatography Mass Spectrometry (LCMS)
Objective
The objective of this work was to investigate the chemistry of compounds potentially remaining
on a medical device following a typical cleaning/autoclaving procedure.
Summary of Results
A Surgical Needle Clamp was treated with a solution of commercial surgical instrument cleaner,
rinsed with deionized water, and then sterilized by autoclaving. The Surgical Needle Clamp was
then submerged in distilled methanol in order to extract any chemical components retained from
the cleaning solution. The methanol extract was concentrated and subjected to analysis by
QTOF-LCMS for identification of residual cleaning components. In addition, the surgical
instrument cleaner solution was analyzed directly by QTOF-LCMS for identification of its
components.
The methanol extract of the Surgical Needle Clamp, collected following cleaning, was found to
contain not only residual components from the surgical instrument cleaner, but also other unique
chemical components. These results suggest that while currently-accepted reprocessing
procedures may be adequate for disinfecting and sterilizing reusable medical devices, further
steps may be necessary for removal of residual chemical components introduced by the
reprocessing procedure itself.
Page 2 of 35

Background
Reusable medical devices are those which can be reused for diagnosis and treatment of multiple
patients. After each use, a reprocessing (cleaning) procedure is followed for
disinfection/sterilization of said device. The procedure is typically carried out by treatment with
a surfactant based detergent followed by sterilization. Cleaning is necessary for the removal of
tissue and other contaminants between patient uses. The reprocessing procedure aims to
adequately remove biological contaminants from a given reusable medical device as effective
sterilization requires clean surfaces.1
The reprocessing procedure also includes a rinsing step to remove residues of any chemical
agents employed during reprocessing. This rinsing step is of particular importance, as failure to
remove residual cleaning agents and liquid chemical germicides can interfere with subsequent
steps of the reprocessing procedure, resulting in incomplete disinfection/sterilization of the
medical device. Failure to remove residual cleaning agents can also cause damage to a medical
device. However most importantly is the fact that the components of such cleaning agents are
often toxic, and thus their residues pose a health hazard when used on new patients. 2,3
Due to the aforementioned risks associated with incomplete removal of chemical cleaning
agents, it is important to assess the effectiveness of the washing step typically employed during
reprocessing of medical devices. The goal of this analysis was to investigate the chemistry of
compounds potentially remaining on a reusable medical device following a typical reprocessing
procedure. 4
Individual Test Results
A summary of the individual test results is provided below. All accompanying data, including
spectra, has been included in the data section of this report. A summary of the results from direct
analysis of the surgical instrument cleaner solution is given in Table 1, along with those of the
methanol extract of the “reprocessed” Surgical Needle Clamp.
Sample Preparation
A Surgical Needle Clamp was submerged in a large vessel containing 100 mL of a commercial
surgical instrument cleaner. The surgical instrument cleaner solution was prepared at a
concentration of 0.2% (v/v) following the manufacturer’s instructions. The vessel containing the
sample and the cleaning solution was placed in an ultrasonic bath for 1 hour. Following
1
ASTM D7225-13, Standard Guide for Blood Cleaning Efficiency of Detergents and Washer-Disinfectors, ASTM
International, West Conshohocken, PA, 2013, www.astm.org
2
http://www.fda.gov/downloads/medicaldevices/deviceregulationandguidance/guidancedocuments/ucm253010.pdf
3
http://www.halyardhealth.com/media/1514/cleaning_reusable_devices.pdf
4
ASTM F2459-12, Standard Test Method for Extracting Residue from Metallic Medical Components and
Quantifying via Gravimetric Analysis, ASTM International, West Conshohocken, PA, 2012, www.astm.org
Page 3 of 35

sonication, the sample was removed from the cleaning solution and was rinsed with 1L of
deionized water for 30 minutes.
The rinsed sample was sterilized in an autoclave at 121°C for 30 minutes. The sample was then
extracted with distilled methanol (200 mL). This extract was collected and concentrated to a
final volume of 1 mL in a vacuum-assisted centrifugal concentrator.
A second extraction was performed with methanol (2nd
Extract) after the initial cleaning and
initial methanol extraction on the Surgical Needle Clamp. This extraction was performed using a
fresh aliquot of distilled methanol (200 mL) followed by concentration to 1 mL.
Finally, a portion of the surgical instrument cleaner solution (10 mL) was subjected to autoclave
sterilization at 121°C for 30 minutes.
LCMS
Background: QTOF-LCMS combines high mass accuracy time of flight mass spectroscopy with
the power of a liquid chromatography separation to provide detailed information about the
elemental composition of unknowns.
The presence of an additional quadrupole mass spectrometer (Q) provides the added capability to
perform fragmentation experiments. This increases the confidence of unknown identification. It
is preferable that a standard of the suspected unknown be analyzed under identical conditions as
the sample. If the fragmentation patterns, high accuracy mass data, isotope patterns and LC
retention times match for the unknown and standard then there is a very high probability that the
identification is correct. It is possible to gain significant information about the structure of an
unknown, even in cases in which standards are not available by using the molecular formula
generation (MFG) algorithms contained in the Mass Hunter qualitative software.
LCMS requires that the molecule of interest be ionized. Thus, data is typically plotted in positive
and negative modes indicating the charge on the ions. Ion formation is accomplished through the
formation of a molecular adduct using a charge carrying species. Typical charge carriers in
positive ion mode include H+
, Na+
, K+
, NH4
+
etc. Thus the observed mass is typically the mass
of the compound plus the mass of the charge carrier.
The nature of the mobile phase and the ionization conditions determine the ions formed. In
negative ion, the loss of hydrogen is generally observed which results in the loss of one mass unit
(1.0078 amu). Other transformations are also possible including dehydration, dimer formation,
etc.
A number of plots are used to aid in interpreting QTOF-LCMS data. This includes Base Peak
Chromatograms (BPC), Extracted Ion Chromatograms (EIC), Extracted Compound
Chromatogram (ECC), Mass spectra (MS) and Product Ion Spectra (MSMS). A BPC is formed
by plotting the most intense ion at a given retention time. This spectrum is particularly useful for
identifying the retention time of unknowns. EICs are formed by plotting a single mass at all
retention times. This could be considered a plot of peak intensity (~compound concentration) for
Page 4 of 35

a single compound (and its isomers) versus retention time. ECC’s are the sum of all the ions
determined to be related to a single compound.
MS spectra plot the observed masses and their intensities at a single retention time. MS/MS
spectra show the fragmentation pattern for a single compound. Mass Spectra plot the mass to
charge ratio (m/z) and not the mass of the compound.
All structures indicated represent best estimates based on the data observed. In most cases the
MS/MS fragmentation spectra have been consulted briefly to aid in identification of possible
structures.
Results
The surgical instrument cleaner solution was first analyzed directly by QTOF-LCMS in order to
determine target compounds which may be observed in the methanol extract of the Surgical
Needle Clamp. Also analyzed directly was a portion of the surgical instrument cleaner solution
which had undergone autoclave sterilization. Comparison of results for this sample and those of
the cleaning solution itself revealed that components of the cleaning solution do not under
degradation as a result of autoclave sterilization.
After the cleaning and extraction of the Surgical Needle Clamp, the concentrated methanol
extract was also analyzed by QTOF-LCMS. Results from these analyses are given in Table 1. It
was found that the most polar compound observed in the cleaning solution, diethanolamine, was
effectively removed during rinsing. However a number of compounds present in the cleaning
solution remain on the needle clamp following the reprocessing procedure. Furthermore, the
methanol extract of the reprocessed needle clamp was also found to contain other unique
components. Results from the analysis of the second extract suggested that even after extraction
with distilled methanol, some surfactants from the cleaning solution still remained on the needle
clamp. Extracted ion chromatograms of the needle clamp extract, surgical instrument cleaner
solution, and control are shown in Figure 1 and Figure 2.
Results of this analysis suggest that not only is the washing step typically employed during
reprocessing of medical devices potentially inadequate for removal of chemical components
from surgical instrument cleaner solutions, but also that a particular medical device may gain
chemical contaminants during the reprocessing procedure. With respect to its aim, the currently-
accepted reprocessing procedure may be sufficient in adequately removing biological
contaminants from a given reusable medical device, but results of this analysis suggest that it can
also introduce new chemical residues. Because of the potential hazards posed by such residues
on reusable medical devices, the reprocessing procedure may need to be modified to include a
more effective washing step for adequate removal of these components.
Page 5 of 35

Table 1
Summary of LCMS Results
RT Positive m/z
Negative
m/z
Mass Best Match Score Diff. Possible ID
Surgical
Instrument
Cleaner
Solution
Needle
Clamp
Extract
0.25 106.0863 105.0790 C4H11NO2 99.93 -0.91
Diethanolamine
X
4.29 288.2534 287.2460 C16H33NO3 99.51 0.2
Lauramide diethanolamine
(Cocamide DEA)
X X
4.38 297.1538 298.1602 C16H26O3S 97.22 -2.71
4-Decyl benzene
sulfonic acid*
X X
4.53 330.2101 311.1691 312.1759 C17H28O3S 98.93 -1.58
4-Undecyl benzene
sulfonic acid*
X X
4.70 325.1846 326.1916 C18 H30 O3 S 98.79 -1.19
4-Dodecylbenzene
sulfonic acid*
X X
4.66 316.2852 315.2773 C18 H37 NO3 98.28 -1.79
Palmitamide Diethanolamine
(Cocamide DEA)
X X
4.84 424.3639** 406.3294 C22H46O6 97.80 -2.02
Dodecyl PEG
X X
Page 6 of 35

Table 1
Summary of LCMS Results
RT Positive m/z
Negative
m/z
Mass Best Match Score Diff. Possible ID
Surgical
Instrument
Cleaner
Solution
Needle
Clamp
Extract
4.84 339.2006 340.2080 C19H32O3S 98.02 -1.7
4-Tridecyl Benzene
Sulfonic Acid*
X X
5.06 496.4218** 478.3870 C26H54O7 94.93 -2.99
Tetradecyl PEG
X X
5.52 550.6304 549.6231 C38H79N 93.76 -3.4
Alkyl amine
X
X-detected
*Compound observed in method blank control at significantly lower abundance
**Ion is a single member of a polymeric series, addition ions are observed
Page 7 of 35

Figure 1 - LCMS Extracted Ion chromatograms, positive ionization
Figure 2 - LCMS Extracted Ion chromatograms, negative ionization
Page 8 of 35

Analysis Conditions
This section of a Jordi report provides information on the methods used including instrument
type, temperatures, solvents, sample preparation, etc. The specific conditions have been removed
for this case study.
Closing Comments
Jordi Labs’ reports are issued solely for the use of the clients to whom they are addressed. No
quotations from reports or use of the Jordi name is permitted except as authorized in writing. The
liability of Jordi Labs with respect to the services rendered shall be limited to the amount of
consideration paid for such services and do not include any consequential damages.
Jordi Labs specializes in polymer testing and has 30 years experience doing complete polymer
deformulations. We are one of the few labs in the country specialized in this type of testing. We
will work closely with you to help explain your test results and solve your problem. We
appreciate your business and are looking forward to speaking with you concerning these results.
Sincerely,
Kevin Rowland
Kevin Rowland, M.S.
Senior Chemist
Jordi Labs LLC
Mark Jordi
Mark Jordi, Ph. D.
President
Jordi Labs LLC
Michael A. Zomoa’
Michael A. Zompa, Ph.D.
Senior Chemist
Jordi Labs LLC
Page 9 of 35

Qualitative Analysis Report
150 0 ESI
Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
User Spectra
Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Column3
Info.
User Chromatograms
Fragmentor Voltage Collision Energy Ionization Mode
Column3
Sample Group
Acquisition SW
Version
Column4
6200 series TOF/6500 series
Q-TOF B.05.01 (B5125.1)
5/5/2015 6:10:14 PM
IRM Calibration Status Success DA Method Default.m
Comment
Instrument Name Instrument 1 User Name
Acq Method NEW-default-Dual-ESI-pos-07MLMIN.m Acquired Time
Data Filename J8906_MtdBlk_pos_1.d Sample Name mtdblk
Sample Type Blank Position P1-B1
Page 1 of 2 Printed at: 2:21 PM on: 5/15/2015
Page 11 of 35

--- End Of Report ---
Page 12 of 35

150 0 ESI
Column3
Info.
User Chromatograms
Column3
Sample Group
Acquisition SW
Version
Column4
Q-TOF B.05.01 (B5125.1)
5/5/2015 7:17:45 PM
Comment
Acq Method NEW-defult-Dual-ESI-neg-07MLMIN.m Acquired Time
Data Filename J8906_MtdBlk_neg_2.d Sample Name mtdblk
Page 13 of 35

150 0 ESI
Mass
287.2469
Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Score (DB)
97.25
Hits (DB)
5
Difference
-0.75
Database Results
m/z
288.2541
Name
cocamide diethanolamine
Formula
C16 H33 N O3
User Spectra
Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Column3
Info.
User Chromatograms
Column3
Sample Group
Acquisition SW
Version
Column4
Q-TOF B.05.01 (B5125.1)
5/5/2015 9:36:21 PM
Comment
Data Filename J9714_CleaningSoln_pos_1.d Sample Name CleaningSoln
Page 14 of 35

Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Diff.
-1.78
-0.96
-1.4
Ion Form.
C17 H29 O3 S
C17 H28 Na O3 S
C17 H32 N O3 S
Best Match
C17 H28 O3 S
C17 H28 O3 S
C17 H28 O3 S
Score
91.89
98.84
98.44
MFG Results
m/z
313.1834
335.1655
330.2101
Mass
312.1765
312.1762
312.1764
Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Score
99.93
Diff.
-0.91
Ion Form.
C4 H12 N O2
MFG Results
m/z
106.0863
Mass
105.0791
Best Match
C4 H11 N O2
Page 15 of 35

Score
96.67
Diff.
-2.6
Ion Form.
C22 H50 N O6
MFG Results
m/z
424.3642
Mass
406.3305
Best Match
C22 H46 O6
Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Score
94.93
Diff.
-2.99
Ion Form.
C26 H58 N O7
MFG Results
m/z
496.4224
Mass
478.3884
Best Match
C26 H54 O7
Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Score
98.28
Diff.
-1.79
Ion Form.
C18 H38 N O3
MFG Results
m/z
316.2851
Mass
315.2779
Best Match
C18 H37 N O3
Page 16 of 35

150 0 ESI
Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Score
99.27
Diff.
-1.15
Ion Form.
C16 H25 O3 S
MFG Results
m/z
297.1534
Mass
298.1606
Best Match
C16 H26 O3 S
User Spectra
Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Column3
Info.
User Chromatograms
Column3
Sample Group
Acquisition SW
Version
Column4
Q-TOF B.05.01 (B5125.1)
5/5/2015 10:27:56 PM
Comment
Data Filename J9714_CleaningSoln_neg_1.d Sample Name CleaningSoln
Page 17 of 35

Score
98.02
Diff.
-1.7
Ion Form.
C19 H31 O3 S
MFG Results
m/z
339.2006
Mass
340.2078
Best Match
C19 H32 O3 S
Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Score
98.79
Diff.
-1.19
Ion Form.
C18 H29 O3 S
MFG Results
m/z
325.1846
Mass
326.192
Best Match
C18 H30 O3 S
Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Score
98.93
Diff.
-1.58
Ion Form.
C17 H27 O3 S
MFG Results
m/z
311.1691
Mass
312.1764
Best Match
C17 H28 O3 S
Page 18 of 35

Page 19 of 35

150 0 ESI
Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Score
83.25
Diff.
1.93
Ion Form.
C4 H12 N O2
MFG Results
m/z
106.0862
Mass
105.0788
Best Match
C4 H11 N O2
User Spectra
Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Column3
Info.
User Chromatograms
Column3
Sample Group
Acquisition SW
Version
Column4
Q-TOF B.05.01 (B5125.1)
5/5/2015 7:53:19 PM
Comment
Data Filename J9714_Sample_pos_1.d Sample Name Sample
Page 20 of 35

Score
99.31
Diff.
-0.99
Ion Form.
C17 H32 N O3 S
MFG Results
m/z
330.2099
Mass
312.1762
Best Match
C17 H28 O3 S
Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Score
99.51
Diff.
0.2
Ion Form.
C16 H34 N O3
MFG Results
m/z
288.2534
Mass
287.246
Best Match
C16 H33 N O3
Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Score
93.76
Diff.
-3.4
Ion Form.
C38 H80 N
MFG Results
m/z
550.6304
Mass
549.6231
Best Match
C38 H79 N
Page 21 of 35

Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Score
95.62
Diff.
-0.41
Ion Form.
C22 H50 N O6
MFG Results
m/z
424.3639
Mass
406.3296
Best Match
C22 H46 O6
Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Score
98.26
Diff.
-1.57
Ion Form.
C18 H38 N O3
MFG Results
m/z
316.2853
Mass
315.2778
Best Match
C18 H37 N O3
Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Page 22 of 35

Score
96.88
Diff.
-1.99
Ion Form.
C26 H58 N O7
MFG Results
m/z
496.4219
Mass
478.3879
Best Match
C26 H54 O7
Page 23 of 35

150 0 ESI
Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Score
98.94
Diff.
-1.48
Ion Form.
C16 H25 O3 S
MFG Results
m/z
297.1535
Mass
298.1607
Best Match
C16 H26 O3 S
User Spectra
Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Column3
Info.
User Chromatograms
Column3
Sample Group
Acquisition SW
Version
Column4
Q-TOF B.05.01 (B5125.1)
5/5/2015 8:44:52 PM
Comment
Data Filename J9714_Sample_neg_1.d Sample Name Sample
Page 24 of 35

Score
96.8
Diff.
-2.82
Ion Form.
C18 H29 O3 S
MFG Results
m/z
325.1853
Mass
326.1925
Best Match
C18 H30 O3 S
Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Score
97.63
Diff.
-2.36
Ion Form.
C17 H27 O3 S
MFG Results
m/z
311.1695
Mass
312.1767
Best Match
C17 H28 O3 S
Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Score
97.11
Diff.
-2.24
Ion Form.
C19 H31 O3 S
MFG Results
m/z
339.2007
Mass
340.208
Best Match
C19 H32 O3 S
Page 25 of 35

Page 26 of 35

150 0 ESI
Column3
Info.
User Chromatograms
Column3
Sample Group
Acquisition SW
Version
Column4
Q-TOF B.05.01 (B5125.2)
4/27/2015 7:10:15 PM
Comment
Data Filename J8906_PreAutoTest_pos_2.d Sample Name mtdblk
Sample Type Blank Position P1-D1
Page 27 of 35

150 0 ESI
Column3
Info.
User Chromatograms
Column3
Sample Group
Acquisition SW
Version
Column4
Q-TOF B.05.01 (B5125.2)
4/27/2015 7:45:55 PM
Comment
Data Filename J8906_PreAutoTest_neg_1.d Sample Name mtdblk
Sample Type Blank Position P1-D1
Page 28 of 35

150 0 ESI
Score
99.37
Diff.
-1.28
Ion Form.
C13 H17 O2
MFG Results
m/z
205.1236
Mass
206.1309
Best Match
C13 H18 O2
User Spectra
Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Column3
Info.
User Chromatograms
Column3
Sample Group
Acquisition SW
Version
Column4
Q-TOF B.05.01 (B5125.1)
5/23/2015 12:58:20 AM
Comment
Data Filename ibustandard_neg.d Sample Name Standard
Sample Type Blank Position P1-F9
Page 29 of 35

150 0 ESI
Score
99.76
Diff.
-0.25
Ion Form.
C13 H22 N O2
MFG Results
m/z
224.1645
Mass
206.1307
Best Match
C13 H18 O2
User Spectra
Fragmentor Voltage
150
Collision Energy
0
Ionization Mode
ESI
Column3
Info.
User Chromatograms
Column3
Sample Group
Acquisition SW
Version
Column4
Q-TOF B.05.01 (B5125.1)
5/23/2015 12:42:19 AM
Comment
Data Filename ibustandard_pos.d Sample Name Standard
Sample Type Blank Position P1-F9
Page 30 of 35

150 0 ESI
150 0 ESI
150 0 ESI
Column3
Info.
User Chromatograms
Column3
Sample Group
Acquisition SW
Version
Column4
Q-TOF B.05.01 (B5125.1)
5/23/2015 4:02:38 AM
Comment
Data Filename J8906_1xCleanSolnAutoClave_neg_1.d Sample Name 1xCleanSolnAutoClave
Sample Type Blank Position P1-C7
Page 31 of 35

150 0 ESI
150 0 ESI
Page 32 of 35

150 0 ESI
150 0 ESI
150 0 ESI
Column3
Info.
User Chromatograms
Column3
Sample Group
Acquisition SW
Version
Column4
Q-TOF B.05.01 (B5125.1)
5/23/2015 3:11:03 AM
Comment
Data Filename J8906_1xCleanSolnAutoClave_pos_1.d Sample Name 1xCleanSolnAutoClave
Sample Type Blank Position P1-C7
Page 33 of 35

150 0 ESI
150 0 ESI
150 0 ESIFragmentor Voltage Collision Energy Ionization Mode
Page 34 of 35

Page 35 of 35

Case Study: Sterilization of Surgical Instruments

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