Development of on-line sample pretreatment system for enhancement of detector’s sensitivity
Yuki Hashi
Shimadzu International Trading (Shanghai) Co., Limited
14/F, Life Tower, No.16 Chaoyang Men Wai Street, Beijing, China 100020
y-hashi@shimadzu.co.jp

Introduction
    Recently, high throughput analysis is the important consideration for development of new analytical methods. Especially, sample pretreatment procedure is the crucial step for high throughput determination. Therefore, several automated on-line sample pretreatment systems coupled with high performance liquid chromatography instruments have been developed. In order to prepare the sample for chromatographic analysis automatically, pretreatment pre-column equipped with high pressure switching valve plays an important role. These pretreatment pre-columns are working for the elimination of co-elutes which may influence the separation and detection, and the concentration of the target compounds to improve the sensitivity. The automated on-line sample pretreatment systems enable us not only to minimize the sample preparation time, but also to obtain acceptable analysis precision. However, these systems must have a reasonable recovery and carry over capability to the target samples, which are most important factors to be validated. We have developed on-line sample pretreatment systems with novel dilution function in order to load a large amount of sample for high sensitive detection.
Result and Discussion
   The dilution line is installed after the sample injection device to dilute the sample with proper dilution solvents for better extraction efficiency (Figure 1). The diluted sample is then effectively trapped by the pre-column regardless the sample loading amounts as well as sample solvents. This system can be used for cleaning validation in pharmaceutical application. For cleaning validation, the sample is extracted by 100 % organic solvent such as ethanol, and the sample is injected to HPLC system. For large volume injection for high sensitive detection, peak shape of the target sample is deteriorated when dilution line is not installed (Figure 2). On the other hand, in case of using dilution line, regardless the sample amount, no peak deterioration is observed.

   LC-MS is an effective and efficient method for the determination of drugs in biological samples such as plasm. Efficient sample pretreatment, for example deproteinization, is critical to achieve reliable results as well as to maintain LC-MS performance. Off-line pretreatment methods are often limited in that the procedures are often time-consuming and as a result constrain high throughput analysis. To resolve this issue a number of pretreatment columns have been applied to achieve robust and reliable system performance whilst still providing high recoveries for on-line pretreatment system. A novel methylcellulose - immobilized reversed-phase pretreatment column (Figure 3) is newly developed to improve recovery and durability for API-MS detection. In this case, the diluting line is contributing to enhance the recovery yield (Table 1).

   The column material has an outer surface with immobilized methylcellulose with an inner surface of conventional ODS. As the column particle size is 50um it also provides minimal back pressure and a higher tolerance to biological materials.
In the field of impurity profile study in pharmaceutical research as well as identification of unknown compounds in natural products research, this dilution line technique together with LC-MS is useful in order to remove phosphate buffer for connection of LC-MS instrument since most of LC applications have been obtained by phosphate buffer conditions which may not be applicable to LC-MS (Figure 4). Sample selection loop installed in the switching valve 1 is functioning to fractionate the target peak compound separated under phosphate buffer conditions. The trapped compound is transferred to sample trap column. However, it may be difficult for this column to trap the target compound in case of using ODS trap column if the mobile phase contains high concentration of organic solvent. In order to enhance this trap efficiency of the sample trap column, dilution line is installed after the switching valve 1. Thus, the target compound contained high concentration of organic solvent as well as phosphate buffer is diluted by pure water delivered by dilution pump. Therefore, the trap efficiency of the sample trap column is increased. And also, phosphate buffer is removed since the pure water is used to trap the target compound. Finally, the target compound is detached by organic solvent to identify the target compounds using API-MS instrument.

Conclusion
    The dilution line is contributing to improve the trap efficiency in case of large volume injection for automated sample pretreatment system. Therefore, higher sensitivity as well as better recovery yield can be obtained. This line can be applied for pharmaceutical applications as well as natural product research field.