Application Note: AN 109D

Analysis of Stability Over 5000 Injections of Testosterone and 17Hydroxyprogesterone on IONICS 3Q 220 Triple Quad Mass Spec
2.1 Mass Spectrometry Conditions

Quick Facts:>5000 injections were performed.

Liquid Chromatography: Shimadzu
Prominence UFLC

Mass Spectrometry: IONICS 3Q 220
triple quadrupole mass spectrometer

ESI Source

CV <3.2% in final 1200 injections

An IONICS 3Q 220 equipped with the Torrent MultiOption Dual Source™ interface with both probes setin ESI mode was used to perform the analysis of bothtestosterone and 17-hydroxyprogesterone.
Below, Table 1 outlines the instrument settings usedduring the method.

1. Introduction
Robustness and stability are important considerations
when choosing a mass spectrometry platform.
Instruments with reduced cleaning and maintenancerequirements are able to be run for longer periods oftime enabling greater laboratory efficiency. LC-MS/MS
continues to be the platform of choice when highperformance, high throughput and high sensitivity arerequired. Already accepted in big pharma,
environmental and food testing labs for small
molecule analysis, new applications in clinicalchemistry labs are increasing the demand forsimplified, easy to use systems.
IONICS 3Q triple quadrupole mass spectrometers aredesigned to maximize uptime by utilizing a variety ofinnovative features. This experiment, conducted on
an IONICS 3Q 220 illustrates the day-after-day
robustness of the instrument. Over the course of thisexperiment more than 5000 injections of testosterone
and 17-hydroxyprogesterone in human serum wereanalyzed with remarkably consistent, high quality
results.

2. Method

Table 1: Instrument Settings
ESI Voltage (Volts)
HSID Temp. (oC)
Nebulizer Gas Setting
Drying Gas Setting
Heating Gas Setting
Source Temp. (oC)
Dwell Time (ms)Pause Time (ms)50002252001202502501005The Q1 and Q2 mass filters were set to unit
resolution. For each compound, two MRM transitions
were monitored: m/z at 289.2/97.1 and 289.2/109.1
for testosterone as well as m/z at 331.2/109.1 and331.2/97.1 for 17-hydroxyprogesterone.

2.2 LC Conditions
The separation was performed on a Shimadzu UFLC
system (including two pumps, an autosampler, adegasser, and a column oven). 1µL of the sample wasloaded on a Imtakt Cadenza CD-C18 HT column(50x2mm, 3µm) at 40 °C. The total LC cycle time was3 min. Solvent A was composed of 5% methanol inwater with 0.1% formic acid and 5 mM ammonium
acetate. Solvent B was composed of 5% water inmethanol with 0.1% formic acid and 5 mM
ammonium acetate. The flow rate was 450 µL/min.

Testosterone and 17-hydroxyprogesterone werepurchased from Sigma (Milwaukee, WI) and used
when received. The two compounds were spiked inprotein precipitated human serum. The injection
concentration used was 1 ng/mL. All solvents usedwere HPLC grade.

Global Headquarters: 32 Nixon RD, Unit 1, Bolton ON, L7E 1W2, Canada. Email: info@ionics.ca, Phone: 1-905-857-5665, Fax: 1-905-857-3040, www.ionics.ca

For Research Use OnlyApplication Note: AN 109D

3. Results
3.1 Extracted Ion Chromatogram (EIC)

LC-MS/MS
EICsoftestosterone
and17hydroxyprogesterone in protein precipitated humanserum are shown in Figures 1a and 1b. More than
5000 injections were completed within a week. The
performance and calibration of the 3Q 220 instrument
remained stable and maintained very low CVs.

Figure 2: Over the final 1200 injections oftestosterone and 17-hydroxyprogesterone, theinstrument maintained CVs of 2.3 and 3.2%
respectively an indication that despite the highvolume of samples the instrument had run, results
remained remarkably consistent.

8e+417 Hydroxyprogesterone, CV% < 3.2

Figure 1(A): LC-MS/MS EIC of testosterone inprotein precipitated human serum.

Peak Area

6e+44e+4Testosterone, CV% < 2.3
2e+400200

400

600

8001000

Injections

4. Conclusion
Time (Minutes)

Figure 1 (B): LC-MS/MS EIC of 17hydroxyprogesterone in protein precipitated humanserum.

After more than 5000 injections of testosterone and17-hydroxyprogesterone in human serum, the 3Q 220
maintains excellent performance. The CVs for
testosterone and 17-hydroxyprogesterone were 2.3%
and 3.2% respectively demonstrating exceptional
reproducibility of results over a long period. With easeof maintenance, unmatched stability and the ability torun for an extended time without the need forcleaning, IONICS 3Q 220 is perfectly suited to meetthe needs of high volume laboratories.

5. Contact Information
To learn more about IONICS Mass Spectrometry, ourproducts or services please visit our website orcontact us directly.
Time (Minutes)

3.2 Reproducibility

For testosterone and 17-hydroxyprogesterone, thechanges of peak area with injections are shown inFigure 2. The data in Figure 2 represents the final
1200 injections (5000 total). The repeated injections
have excellent percent CV’s; 3.2% for 17hydroxyprogesterone and 2.3% for testosterone.

Global Headquarters: 32 Nixon RD, Unit 1, Bolton ON, L7E 1W2, Canada. Email: info@ionics.ca, Phone: 1-905-857-5665, Fax: 1-905-857-3040, www.ionics.ca

For Research Use Only1200