Using ISOLUTE SLE+ for improved vitamin D analysis at Chiba University Hospital

Thank you for giving us your time today. First, please tell us about the background of your research.

Associate Professor Satoh: “The main topic of our research is developing a method for vitamin D analysis and its clinical applications. We are also working on an analysis method for steroid hormones. We are currently installing a new liquid chromatography mass spectrometer for analysis.

In our current clinical studies, we are using antibodies to detect vitamin D. Although this method is faster and cheaper than traditional approaches, there is a risk we might measure various other elements by accident if the concentration of antibodies is too low. We might even be measuring other variants of vitamin D with a different biological activity.

25-(OH)-vitamin D reflects the supply of vitamin D overall in the body. In several countries, this is being measured instead of the active types. There are plenty of manufacturers selling reagents for detecting vitamin D in America, but different reagents and experiments using other reagents yield different results. So we are trying to promote the more robust LC-MS/MS method in Japan.

It allows us to measure individual elements accurately and get useful information for the patient. We may be able to narrow the range referred to as the “grey zone” for diagnostic purposes.”

Ishige: “I am a clinical technologist, and in my field of clinical testing I can see a movement to correct the variance between different methods through standardization. So the LC-MS/MS method, which gets us accurate results, is a very important factor.”
Satoh: “LC-MS/MS is at the top of the available measurement techniques, and has become an absolute standard. So devel- oping measurement methods in this area comes with a huge responsibility.”

Why did you decide to use ISOLUTE SLE+ in your research?

Ishige: “I attended a conference in America in 2013 and saw a poster presentation of ISOLUTE SLE+. I have seen lots of presentations on analysis method for vitamin D before, but the SLE+ poster showed that it could decrease ion suppression as well, which caught my attention. I knew it would become an obstacle at some point, so I got interested and wanted to try it.”

Satoh: “After returning to Japan, Ishige-san ordered them for us. We then met some people from Biotage at an academic meeting here in Japan. Things moved pretty fast from there.”

I understand you had tried many methods before ISOLUTE SLE+. How is ISOLUTE SLE+ different?

Satoh: “Before ISOLUTE SLE+, we used protein precipitation for extraction. But it was difficult to remove contaminants with that technique.”

Ishige: “Another big advantage was that we could use derivatizing agents with ISOLUTE SLE+. Our aim was to develop a robust method with a streamlined workflow. With protein precipitation, the collected sample contains water, which stops the derivatization from working, and makes it difficult to get any measurements. Evaporating the sample after protein precipitation would allow us to derivatize, but the evaporation step negates the advantage of being able to analyze directly after sample preparation.”

Satoh: “When thinking about tests and applications, the balance between required sensitivity and workload becomes important. If the sample doesn’t dry up after protein precipitation, there is always some water left. But with ISOLUTE SLE+, we can remove all the water and use an organic solvent. As a result, we are now able to measure and include the derivatization process without a problem. This was very helpful.”

Ishige: “With a low concentration compound, we could have increased the amount of sample, but that also means the number of contaminants will increase. It is better to use a smaller sample from the patient.”

Satoh: “ISOLUTE SLE+ is also very easy to use, I think that’s the biggest advantage. All you have to do is load the sample at the top and collect the liquid that comes off from the bottom. With solid phase extraction, we had to balance out the conditions, insert the sample, wash it, and then elute. There are too many steps. With ISOLUTE SLE+, all there is to do is load the sample and move on to the elution process. So, the sample preparation is over in no time.”

You are also using PRESSURE+96 and SPE Dry 96 from Biotage. What are you thoughts on them?

Satoh: “I couldn’t do my job without these instruments (laughs). In the sample prep process, it is standard to dry the samples under nitrogen gas, or the sample might oxidize. I can’t skip this procedure, so I always felt a need for an evaporation system that handles sample 96- well plates. Biotage had the ISOLUTE SLE+ and other equipment for vitamin D analysis, so I ordered both.”

So what is on your wish-list?

Satoh: “There are a lot application notes out there already, but I would like to see more in the future. We would also like information on the methods that don’t work. Application notes only show the successful cases (laughs). The extraction process for vitamin D worked well when I followed the steps in the application note, but I am not so sure about the steroids I am testing now. It takes time to back-track what went wrong when a test fails, so it would be great if I could find information on failed cases as well.

Although Biotage is a foreign company to us, they are convenient to work with because you guys provide information very quickly. The other day I received some posters from you, it seems the regional staff had just contacted the application centre in England and requested them for us. Other foreign manufacturers sometimes give responses like, “We asked headquarters, but it couldn’t be done”. Biotage provides me with all the information quickly. That builds trust.

Chiba University Hospital
Chiba University Hospital had its origins in the Kyoritsu Hospital (near the current Chiba Shrine), established in 1874 through donations from volunteers in Chiba, Noborito, and Samukawa.
All through the years, the hospital has scored many achievements in cutting-edge research, and produced many medical professionals who have lead the world.
Chiba University Hospital is undertaking a variety of initiatives to develop safe and reliable medical care while respecting the wishes of patients and to cultivate outstanding medical professionals who will lead the way to the future.

• Site area 84,699m²
• Total floor area 121,944m2
• Number of beds 850
•  Number of outpatients 2,134 (persons/day) 

Literature number: PPS431