Jessica+Sima-+final+paper


 * __Current LC-MS/MS techniques used for the detection of testosterone levels in serum samples__**


 * Abstract:**

The current practices of testosterone detection with the used of immunoassays does not provide the complete sensitivity range that is needed for the low circulating serum levels found in women and children. The LC-MS/MS techniques that are becoming the have lowered the limit of detection and eliminated human error in assays. The increased accuracy is resultant from the coupled use of protein precipitation and solid-phase extraction which removes possible causes of matrix interference and increases the sample concentration. When run in tandem with a mass spectrometer with an APCI interface, the reliability of the method is made even stronger. As a method that is more cost effective per sample run and allows for an increase in throughput, LC-MS/MS is becoming the gold standard for testosterone detection.


 * Introduction:**

Testosterone (TE), shown in Figure 1, plays a large role in many physiological processes such as the development of the male phenotype, growth, metabolism, and bone remodeling (Kushnir, 2006). It is found in both males and females, though the levels in females are drastically lower than that found in males. Female serum levels are typically circulating in concentrations that are about five percent of those found in males (Kushnir, 2006).Due to the many roles that TE plays in development there are many needs for detection of circulating levels. As the major androgenic hormone in humans, TE is measured to diagnose clinical conditions either in excess or deficiency (Ravinder, 2008). The use of TE detection is also utilized in doping control which has become more prevalent in the recent decades.

An issue with TE detection arises when attempting to measure serum levels females or children. Immunoassay techniques commonly used have a lower limit of detection (LOQ) of approximately 50 ng/mL, a level that is not sensitive enough to detect the circulating levels of TE in women and children (Ravinder, 2008). Another prevalent techniques used for steroid detection is gas chromatography with tandem mass spectrometry (GC-MS). However, the use of GC-MS requires a time consuming hydrolysis of the analytes that are sought after. Table 1 shows a comparison between immune assay technology and current practices with LC-MS/MS (Vogeser, 2007).



With high-pressure liquid chromatography tandem mass spectrometry (HPLC-MS/MS), sample preparation time is greatly reduced, the troubles with quantitative recovery associated with hydrolysis are eliminated, and there is an increase of flexibility in steroid metabolism studies (Borts, 2000). LC-MS/MS is a reliable assay for both clinical and research purposes, where androgen measurement in women is required (Janse, 2007). Using liquid-chromatography coupled with mass spectrometry helps to lower the LOQ which is necessary when trying to evaluate pharmacokinetic studies. To evaluate low oral doses of TE it was necessary to improve the LOQ to 25 pg/mL (Dams, 2003). This brings up a benefit versus cost argument; it is questioned whether the sensitivity of LC-MS/MS techniques is worth the cost of the equipment required and the trained staffed to run the samples. One must evaluate the cost versus benefit of sample preparation, analysis time, and sensitivity that arise from the use of LC-MS/MS. Pharmaceutical companies are among the most hit when this argument arises, they are constantly under pressure to reduce development times, which is also in conjunction with an increase of samples that requiring pharmacokinetic (PK) analysis and a significant decrease in the LOQ (Xu, 2007).

Another problem that arises with the use of serum sample is matrix interference which is made the most prominent when trying to reduce the LOQ needed for TE detection. Matrix interference is defined as the effect of co-eluting residual matrix components on the ionization of the target analyte. Typically, suppression or enhancement of analyte response is accompanied by diminished precision and accuracy of subsequent measurements (Dams, 2003). This interference presents itself with either ion suppression or enhancement and is known to be dependent on the compound being analyzed, the matrix that is present, and the ionization mode used with the mass spectrometer (Xu, 2007). When evaluating the experimental method used it is important to take note of the experimenter’s account for possible matrix interference. By ignoring the effect brought on by a possible matrix effect there may be an adverse effect the reliability of determination of analyte concentrations and the integrity of data generated (Matuszewski, 2003). Matuszewski et. al. found that matrix interference is highly dependent on the mechanism of ionization in the HPLC-MS interface and that under otherwise identical conditions the use of electro spray ionization (ESI) showed significantly greater levels of matrix interference. It is suggested that the increased prevalence of matrix interference with ESI as opposed to that seen with atmospheric-pressure chemical ionization (APCI) has to do with the actual process of ionization. APCI is based on gas phase reactions and ESI is mainly based on liquid-phase reactions (Dams, 2003). Less ion suppression is seen with APCI because unlike with the use of ESI, competition between analytes to enter the gas phase is not present, due to the fact that with APCI neutral analytes are transferred into the gas phase by vaporizing the liquid in a heated gas stream (Van Eeckhaut, 2009).

All the aforementioned issues with TE detection must be taken into account when critically analyzing the experimental methods used. When trying to determine the ideal method sample preparation, the overall time of analysis, the role of matrix interference, and the sensitivity of the assay should all be evaluated.


 * Experimental Techniques:**

//Sample Preparation-//

Recently the most popular sample preparation technique has become solid-phase extraction (SPE). A push for SPE began when laboratories were pressured to use less organic solvent (Marie-Claire, 1999). A key principle for the use of solid-phase extraction is developing a method that is as selective as possible to the analyte of interest. New technologies have made it possible to complete SPE either off-line, before injection into the chromatography column, or on-line which occurs along with injection onto the chromatography column and eliminates human error in the handling of the samples between steps. Along with reducing risk of contamination, the on-line SPE process also helps to eliminate sample loss due to evaporation between subsequent steps. With the use of LC-MS/MS on-line coupling with SPE is made rather simple, due in part to good compatibility of the LC aqueous mobile phases with the SPE of biological samples which are mainly aqueous (Marie-Claire, 1999). Another major benefit of SPE is that it provides increased sample concentration but still may not be the most cost effective process (Xu, 2007).

Protein precipitation (PPT) through the use of an organic solvent is also a very prevalent sample preparation technique mainly due to its simplicity. This practice is widely used in both bioanalytical research and pharmaceutical PK studies for unknown samples (Xu, 2007). After PPT supernatant samples are readily injected onto the chromatography column which makes this sample preparation technique desirable. A risk that arises when simply using PPT as means for sample preparation is that salts and other endogenous material are still present in the supernatant sample which may be the cause ion suppression or enhancement that ultimately leads to increases variation between samples and lack of reproducibility (Xu, 2007). Pitfalls to both SPE and PPT arise from cross contamination of samples during transfer steps (Kushnir, 2006). Like with SPE contamination of samples can be eliminated with the use of automated systems such as Tecan® robotic titration systems, as well as the use of on-line extraction techniques.

//Determination of Matrix interference-//

Due to the increased need high sensitivity in TE assays, a large amount of co extracted compounds could potentially be the cause for background noise and affect the overall method accuracy and sensitivity; phospholipids, found in blank serum, have been cited as a major cause of ion suppression (Kushnir, 2006). Because of this occurrence, the most important step in determining the reliability of the LC-MS/MS methods utilized is to determine if and to what extent does matrix interference play a role in the concentrations of both standard and unknown samples. This feat is accomplished by running a secondary experiment using a chromatography infusion pump that consistently injects a steady concentration of the desired analyte onto the chromatography column. At the same time samples of blank matrix are injected onto the column at the same time as the infusion pump is running. This experiment can be set up in two possible ways which are shown in Figure 2 (Van Eeckhaut, 2009).

An important note to make about the matrix interference experiment is that the steady state concentration of analyte is run higher than the LOQ determined in the original analysis of samples (Van Eeckhaut, 2009). A sample of the resultant data from such an experiment can be seen in Figure 3 (Wang, 2007). In the figure the sample analyte was run in conjunction with three separate serum samples. In the third serum sample that was run a significant dip is apparent on the readout from the infusion pump which indicates ion suppression at that time point in the chromatography method. Since the dip coincides with the elution time of one of the analyte ions the lack of concentration detection is concluded to have come from the suppression caused by the blank serum.



Co-eluting matrix components should be equal for both the internal standard (IS) as well as the analyte in order to maintain a consistent peak-area ratio to be used in the calculations of concentrations. Which means that while the absolute response may be affected, since the analyte to IS peak area ratio remains constant and unaffected the bioanalytical method should still be considered accurate and precise (Wang 2007).

Though matrix interferences are for the most part always present in serum samples, it is common practice to account for their effects by adjusting the chromatography method utilized in order to prevent the co-elution of competing compounds. Long isocratic or gradient chromatographic programs are typically used to ensure that a matrix effect will not present itself at the same retention time for the analyte of interest (Xu, 2007). By preventing a compound from eluting to early off the chromatography column, the experimenter helps to ensure that the ion suppression that usually accompanies early-eluting compounds doesn’t play a role in method accuracy.

//Internal Standard Selection-//

Yet another important technique in method validation I finding the appropriate IS standard to run with the selected analyte. One of the limitations when trying to develop a method for testosterone detection is settling on an internal standard since there is no universally recognized standard in place (Rosner, 2007). As of now the practice in place is to use a stable isotope labeled internal standard, more specifically deuterium labeled testosterone. The standard should be added pre-treatment in order to detect and correct for analyte losses during sample pre-treatment along with any interfering matrix effect (Xu, 2007). One should also take caution when employing the use of a deuterium labeled isotope. According to Wang et. al. some deuterium labeled compounds exhibit unexpected behavior that can play a large role in method validation. A source of this unexpected behavior may arise from deuterium slightly altering the lipophilicity of the analyte which can in turn affect the retention time of the IS in reversed phase chromatography (Wang, 2007). The overall expectation is to reduce matrix interference with the deuterium labeled isotope.

//Choosing the appropriate chromatography column-//

The columns utilized for high-throughput detection are short columns with small densely packed particles which are run with gradient chromatography to reduce the time needed to complete the analysis of each individual sample (Xu, 2007). Columns are either ultra-pressure liquid chromatography (UPLC) or HPLC capable. The use of a UPLC column can offer higher resolution, speed, and sensitivity. Wang et. al. reported that UPLC-MS produced anywhere from a fifty to a one hundred percent increase in sensitivity and at the same time reduced the cycle time when compared to HPLC-MS. HPLC still remains the standard for liquid-chromatography.

A way of speeding up HPLC cycle time while also increasing resolution is to us a monolithic column. The structure of monolithic columns supports and increased flow rate of mobile face without creating significant back pressure on the injection system. A characteristic of such columns is that they are made up of porous silica particles that form macropores that allow for high external porosity (Xu, 2007). Because of this structure and functionality, monoliths can be linked in series to allow for an increase in throughput. An approach to using monolithic separation is to combine it with high-flow on-line extraction to increase both throughput and sensitivity of the assay (Xu, 2007). A major drawback to the use of monolithic chromatography is that it demands an increase in the amount of organic solvent used, which has already created a debate in laboratory practices.

//Increasing Sensitivity-//

By using on-line extraction techniques, assay sensitivity is increased greatly. It has been shown that when coupled together, on-line SPE and LC-MS/MS improves sensitivity and lowers the LOG without a drastic shape in peak shape or resolution (Li, 2005). At the same time, an increase in the amount of analyte injected onto the column corresponds to an increase in sensitivity of the assay. Experiments have shown that the increase in the analyte injected is linearly proportional to the sensitivity detected (Li, 2005). A risk to increasing the flow rate is that it becomes possible to over saturate the column and the detector. By over saturating the column an issue of carry-over is brought about. Residual compound left on the column elutes along with the following sample injection and provides a false increase in the detection level of the analyte. By over saturating the detector there is a resultant loss in sensitivity. The high standards injected into the system will measure lower than the actual value and the range between the high and low standard will be reduced. Choosing between APCI and ESI ionization modes also plays role in increasing sensitivity of an assay. ESI tends to have a high sensitivity (Rybak, 2008) but has also been linked to high occurrence of matrix interference. Müller et. al. found that the fear of matrix interference shouldn’t prevent the use of ESI methods, and that the greater sensitivity produced outweighs the extra steps needed to eliminate the role of matrix interference (2002). In fact, when trying to determine the testosterone levels found in the fecal matter of Cebus capucinus, a species of primate, ESI mode was utilized to get sensitivity that produced an LOQ far below that needed to detect circulating levels (Weltring, 2012). Choosing between ionization modes becomes dependent on the properties of the analyte in question. To find the best suited ionization mode see Figure 4 (Thurman, 2001).




 * Standard Practices used in testosterone detection:**

Currently there is no consensus as to which ionization mode works the best for the detection of testosterone. Both ESI and APCI have been cited; ESI for its increased sensitivity and APCI for its lack of matrix effect. Though no one ionization mode is cited as the best, APCI is the generally accepted method utilized in pharmaceutical PK studies (Dams, 2003). Figure 5 shows the common modes used for the detection of anabolic agents in doping (Thevis, 2007).



Sample preparation techniques include both PPT and SPE, but the best results based on sensitivity and lack of matrix interference arises from the combination of the two techniques on-line with chromatography (Xu, 2007). The combination of the two methods allows for easy purification of the samples and prevents human error by putting it on-line with chromatography. When utilizing urinary samples even further sample preparation is Add Caption required because of the increased presence of endogenous compounds and the decrease level of testosterone (Borts, 2000). Figure 6 shows the mass spectrometer reading of testosterone after the use of on-line SPE-PPT coupled with LC-MS/MS (Ravinder, 2008). The results indicate that this method of sample preparation is sensitive, accurate, and precise assay for serum testosterone. Data shows that the removal of endogenous compounds by the combined method of sample preparation along with more efficient HPLC separation provide the means for a precise and accurate measure of testosterone levels (Matuszewski, 1998).



The use of LC-MS/MS has proven to have much greater sensitivity for testosterone detection than commonly used immunoassays. What is needed now is a stronger push into the use of LC-MS/MS. A universally accepted method is needed especially in the area of doping control which has seen the highest interest of lowering the limit of detection. Numerous findings of the misuse of anabolic androgenic steroids, natural steroid hormones (e.g., testosterone) or designer steroids, as well as other anabolic agents such as the β2-agonist clenbuterol have played a heavy role in all areas of athletics in recent year which has resulted in a push for faster throughput and greater sensitivity (Thevis, 2007).
 * Where to go from here:**

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