JP2023025288A - Automatic pretreatment apparatus for test specimen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To develop a highly versatile automatic pretreatment apparatus for test specimens that can be used for instrument analysis and the like.

SOLUTION: An automatic pretreatment apparatus for test specimens adopts mechanisms 3, 7 capable of freely transferring and lifting sample tubes Tu on a predetermined XY plane at the time of performing various treatments such as extracting a predetermined solvent and the like from the test specimen, and further made it an efficient automatic pretreatment apparatus by moving a pipetting device 9 for sorting and supply of the solution as one unit with the movement of the tubes Tu.

SELECTED DRAWING: Figure 1

COPYRIGHT: (C)2023,JPO&INPIT

Description

There is an application for application of Article 30, Paragraph 2 of the Patent Act. 2) Conference presentation The 112th academic conference of the Japan Society for Food Hygiene (2016), poster presentation P-33, date: October 27, 2016,

The present invention relates to an automatic pretreatment apparatus for test samples. In particular, the present invention relates to an automatic pretreatment device for test samples that is suitably used for instrumental analysis.

Extraction operations are often performed to extract detection targets from test samples, but these operations are often complicated. For example, when analyzing whether or not veterinary drugs, agricultural chemicals, etc. remain, the operation of extracting detection targets such as veterinary drugs, agricultural chemicals, etc. from food test samples or biopsy samples is complicated. There are many.
On the other hand, these operations are often performed according to a predetermined procedure, and if these operations can be automated, data with high accuracy and reproducibility of the extraction operation can be obtained. In addition, there are advantages such as reduced labor costs, uniformity of extraction operations, and elimination of human error.
Thus, it is preferable to automate the pretreatment of test samples used in various analyses, as much as possible.

For example, the inventors of the present application have studied an automatic pretreatment method for mechanically and automatically extracting pesticides for efficient analysis.

JP 2013-3003

However, although the above literature is suitable for the analysis of pesticides, it has little applicability to other analyzes including veterinary drugs. Therefore, we decided to develop a more versatile automatic pretreatment apparatus.

As a result of various investigations under the above-mentioned purpose, as an automatic pretreatment device for test samples, when performing various processing such as extraction of a predetermined solvent etc., a sample tube can be freely moved on a predetermined XY plane. By adopting a transfer mechanism and moving the pipetting device for dispensing and supplying the solution together with the movement of the tube, the device can be made more efficient. , and found that it can be used in automatic pretreatment devices for various instrumental analyses.

In other words, as a result of examining the operability of various extraction processes, in the case of automation, pipetting operations such as collecting the supernatant of the solution in the sample tube and adding the solvent, etc. It has been found that a method of integrating a device for performing measurement with the movement of the sample tube is effective.

That is, the first invention of the present application is
"1) a transfer unit having a movement shaft and a transfer section capable of sliding on the movement axis and moving within a predetermined plane;
2) a tube elevating device that is attached to the transfer section, clamps and releases the sample tube, and is capable of elevating;
3) a vertically movable pipetting device that is attached to the transfer section, has a detachable predetermined tip portion, and sucks or discharges a liquid;
An automatic pretreatment device for test samples. ", is.

Further, in the automatic pretreatment apparatus according to claim 1, the inventors have found that a method of providing a screw cap-type sample tube with a detachable device for the screw cap is effective.
That is, the second invention of the present application is:
"In the automatic pretreatment device, the sample tube is a screw-cap type sample tube, and
4) According to claim 1, the sample tube mounting portion of the screw cap type and a cap attaching/detaching device capable of clamping and rotating the cap portion of the screw cap to attach/detach the cap and place the sample tube on a predetermined place are provided. An automatic pretreatment device for the described test sample. ", is.

Next, the present invention preferably includes a homogenizer mechanism for homogenizing the sample in the sample tube, a tube shaking mechanism, a dispensing device capable of dispensing a solvent into the tube, and a centrifugal separation device.

That is, the third invention of the present application is
"The automatic pretreatment apparatus further comprises: 5) a movable stock section on which the screw-cap type sample tube is placed and which is slidable;
6) an elevating homogenizer device that can be inserted into the sample tube accommodated in the movable stock unit;
7) a rack for accommodating the sample tube, a shaking device having a shaking section capable of shaking the rack, and 8) a mounting section for the screw-cap type sample tube, and dispensing a solution into the sample tube. a solvent dispensing unit having a dispensing device with a pump section capable of
9) comprising a centrifugal separator for centrifuging the sample tube;
3. The automatic pretreatment apparatus for test samples according to claim 2. ", is.

Provision may then be made for a column to perform said further purification.
That is, the fourth invention of the present application is
"The automatic pretreatment device further
10) The automatic pretreatment apparatus for test samples according to claim 3, which includes a treatment section using a column. ”, is.

Furthermore, as a concentration step, it is also possible to adopt a mode equipped with a centrifugal evaporator.
That is, the fifth invention of the present application is
"The automatic pretreatment device further
11) The automatic pretreatment apparatus for test samples according to claim 4, comprising a centrifugal evaporator device. ”, is.

BRIEF DESCRIPTION OF THE DRAWINGS It is the perspective schematic diagram of the whole automatic pretreatment apparatus of 1st embodiment of this invention, and an enlarged perspective view of a transfer part. BRIEF DESCRIPTION OF THE DRAWINGS It is a top view of the whole automatic pretreatment apparatus of 1st embodiment of this invention. FIG. 3 is a perspective view of a transfer unit (transfer section, moving shaft, fixed rail); FIG. 2 is a perspective view of the sample tube lifting device ((1) rising state (2) falling state). FIG. 4 is a schematic front view showing the operation of the sample tube lifting device; It is a perspective view of a pipetting device ((1) ascending state, (2) descending state, (3) photograph of ascending state, and (4) photograph of descending state). FIG. 4 is an enlarged view of a chip attaching/detaching portion; Perspective view of the tip rack (photograph of (1) without tips and (2) with tips) FIG. 4 is a schematic front view of the process of inserting the tip of the chip into the sample tube. It is a perspective view of a cap removing device ((1) drawing (2) photograph). FIG. 10 is a front view showing the operation of attaching and detaching the cap; Fig. 2 is a perspective view showing a mobile stock section and a group of homogenizer devices; It is a perspective view of the shaking device ((1) photo of approximately horizontal state, (2) photo after rotation, (3) perspective view) It is a perspective view of a solvent dispensing unit ((1) drawing (2) photograph). It is a perspective photograph of the stock part of the solvent (for supplying to the solvent dispensing unit). Pressurization unit (syringe pump part to dispensing unit and syringe part for pressurizing the upper part of the column) It is a perspective view of a fixed stock part ((1) drawing (2) photograph). It is a perspective view of the processing part by column or filtration ((1) Drawing (2) Photograph (3) Overall photograph including solvent stock bottle). 1 is a perspective view of a centrifugal evaporator; FIG.

1 Moving shaft 3 Transfer part 5 Fixed rail 6 Transfer unit 7 Tube lifting device 8 Tube clamping part 8-1 Tube clamping bar 9 Pipetting device 10 Tip tip cover part
11 tip rack 12 tip removal member 13 cap removal device 15 cap rotating/moving unit 16 cap lifting unit 17 cap mounting unit 18 cap gripping bar 21 movable tube mounting unit 22 tube holding unit 23 sliding movable stock unit 25 homogenizer unit 26 Slide cover 27 Shaking device 28 Shaking device tube rack 29 Solvent dispensing unit 30 Solvent supply unit 31 Pressurization unit (syringe unit and syringe pump unit)
31-1 Column pressurizing syringe 31-2 Syringe pump for solvent dispensing 33 Fixed stock unit 35 Centrifuge device 37 Centrifugal evaporator device 39 Tube rack 40 Column processing unit 41 Column mounting unit 43 Column solvent supply unit 44 Column pressure supply Part 47 Solvent stock part 49 Centrifugal evaporator 51 Waste liquid bottle 53 Cold trap 55 Suction pump
Tu screw sample tube
C cap (screw type tube lid)
Ti tip (tip)

A first embodiment of the present invention will be described below. However, it goes without saying that the present invention is not limited to these embodiments.

As shown in FIGS. 1 and 2, the automatic pretreatment apparatus of the first embodiment of the present application is an automatic pretreatment apparatus for test samples,
1) a transfer unit having a movement shaft and a transfer section that slides on the movement shaft and is movable within a predetermined plane;
2) a tube elevating device that is attached to the transfer unit and capable of clamping and releasing a screw-cap type sample tube to elevate it;
3) a vertically movable pipetting device that is attached to the transfer section, has a detachable predetermined tip portion, and sucks or discharges a liquid;
4) a screw cap-type sample tube mounting portion, and a cap removing/attaching device capable of holding and rotating the cap portion of the screw cap to attach/detach the cap, and placing the sample tube at a predetermined location;
5) a movable stock unit on which the screw-cap type sample tube is placed and which is slidable;
6) an elevating homogenizer device that can be inserted into the sample tube accommodated in the movable stock unit;
7) a rack for stock containing the sample tube, a shaking device having a shaking section capable of shaking the rack; a solvent dispensing unit having a dispensing device equipped with a pump unit capable of dispensing
9) a centrifugation device for centrifuging the sample tube;
10) The automatic pretreatment apparatus for test samples according to claim 3, comprising a column-based treatment unit;
11) with a centrifugal evaporator device,
An automatic pretreatment device for test samples.

Needless to say, the present invention is not limited to the first embodiment. FIG. 1 is a schematic perspective view of the entire automatic pretreatment apparatus according to the first embodiment of the present invention. In addition, in FIG. 1, a part of the structure such as a waste liquid bottle and a tube rack is omitted.
Next, FIG. 2 is a plan view of the entire automatic pretreatment apparatus according to the first embodiment of the present invention. A part of the structure such as the transfer unit is omitted.
In addition, various devices and units are connected to predetermined motors, various power units, power sources, etc. as power sources. These descriptions are omitted.

○ Transfer Unit The transfer unit 6 in the first embodiment of the present invention includes a transfer section 3 , a moving shaft 1 and a fixed rail 5 as shown in FIG. is attached to the Both ends of the moving shaft 1 are attached to a pair of fixed rails 5 orthogonal to the moving shaft 1 so as to be slidable.
As a result, the moving shaft 1 moves in the Y-axis direction as shown in FIG. Furthermore, since the transfer section 3 can slide on the movement shaft 1, it can move in the X-axis direction as shown in FIG.
In this way, the moving shaft 1 can move in the Y-axis direction, and the transfer section 3 can move in the X-axis direction on the moving shaft 1, so that it can freely move on the XY plane of FIG. ing.
The transfer unit 3 is equipped with a tube elevating device 7 for clamping and releasing a tube Tu, which will be described later, and a pipetting device 9 for sucking or discharging a liquid.

As a result, the tube lifting device 7 and the pipetting device 9 can move freely within the XY plane shown in FIG. Although not shown in FIG. 3, the transfer unit includes a drive unit for driving the movement of the moving shaft 1 in the Y-axis direction and a drive motor for allowing the transfer unit 3 to slide on the moving shaft 1. is installed.

○Sample Tube Lifting Device In the present invention, a sample to be inspected is accommodated in a sample tube Tu having a screw-type cap C, and various operations such as homogenization and extraction are performed in the tube Tu. Here, various devices are arranged on the XY plane in order to perform various processes on the sample tube Tu (FIG. 2).

The tube Tu is transported along with the movement of the transfer section 3 while being clamped by the sample tube elevating device 7 attached to the transfer section 3 of the transfer unit 6 described above.
Next, after being placed at a predetermined position on the predetermined XY axes, it is necessary to lower the tube Tu by the sample tube lifting device 7 attached to the transfer section 3 in order to supply the tube Tu to various devices. There is

Here, as shown in FIG. 4, the sample tube elevating device 7 is attached to the transfer section 3, and has a cylindrical clamping section 8 provided inside a substantially rectangular parallelepiped housing, which can be raised and lowered. freely set. A known method using a pole screw or the like can be used for the lifting mechanism.

In addition, three sample tube clamping bars 8-1 are provided around the circumference of the columnar clamping part 8, making it possible to clamp and release the upper part of the sample cap C as shown in FIG. It is configured.

In addition, although the tube Tu in the embodiment of the present invention is disclosed as a tube Tu with a screw cap, it is not necessarily limited to this. It is also possible to perform pretreatment operations with the tube in condition. In this case, the device to be provided will differ depending on the form of each tube.

○ Pipetting device As shown in Fig. 5, a vertically movable pipetting device 9 is attached to the transfer unit 3, has a detachable tip (tip), and is capable of sucking or discharging liquid. The pipetting device 9 is attached to the transfer section 3 described above, and is attached adjacent to the sample tube lifting device 7 described above.

In the present invention, there is a description that the sample tube lifting device 7 and the pipetting device 9 are attached to the transfer section 3, but the description of this attachment does not necessarily refer to the transfer section 3 and the sample tube lifting device. It is not intended that the device 7 and the pipetting device 9 be clearly separated as members.
For example, even when these are integrally constructed, they are provided with the transfer section 3, the sample tube lifting device 7, and the pipetting device 9 as referred to in the present invention.

As shown in FIG. 6, the pipetting device 9 is provided with a cylindrical clamping portion inside a substantially rectangular parallelepiped housing, and is set to be able to move up and down. A known method using a pole screw or the like can be used for the lifting mechanism.
The pipetting device 9 has a predetermined fitting portion for the tip Ti as shown in FIG. .

A conduit is provided in the center of the fitting portion of the tip to apply a suction pressure to the inside, and the conduit is configured to communicate with a suction device via a tube or the like. It is configured so that a solution can be sucked inside.

Then, a tip Ti of a predetermined size is attached to the fitting portion at the tip of the pipetting device 9 by pressing, and the tip of the pipetting portion is configured to be able to aspirate the solution in the same manner as a normal Pipetman (registered trademark). It is configured.
That is, the inside is constructed so that the suction pressure can be applied so that the solution can be sucked with the tip Ti attached. Further, by releasing the pressure, the sucked solution can be discharged.

In addition, although the first embodiment of the present invention discloses a type in which a tip Ti is attached, the present invention is not limited to this configuration, and for example, a pipette made of glass or plastic is attached. It goes without saying that the configuration may be such that
Also, in the case of using tips Ti, it is necessary to replace the tips when handling different solutions. For this replacement, a prescribed tip rack 11 shown in FIG. 8 is installed. Thus, the tip Ti at the tip can be replaced.

As for the operation of the pipetting device 9, in the case of supplying or aspirating a solution to a predetermined tube Tu, the cap C is removed while the device 9 is positioned above the target tube Tu. The pipetting device 9 is lowered into the opening of the tube Tu in this state, and the tip is inserted through the opening of the tube Tu. And it is configured to discharge or suck the solution.

In addition, in the above pipetting operation, it is preferable to prevent dripping due to liquid leakage from the tip of the tip Ti at the tip in a state where the solution to be dispensed is aspirated.
Therefore, as shown in FIG. 6(3), when the pipetting device is lifted in a suctioned state and moved through the movement of the transfer unit 3, dripping due to liquid leakage from the tip of the tip Ti will occur. In order to prevent this, it is preferable to move the transfer section 3 while the lower part of the tip Ti tip is covered with the tip tip cover part 10 so that even if the liquid leaks, it can be received.

Next, in the present invention, one of the two layers of the solution may be collected for sample purification or the like. In the case of the two layers, the inside of the tube Tu becomes a two-layer solution, and an interface between the layers may occur.

When aspirating and discharging a solution using the pipetting device 9, in order not to aspirate the interface layer between the layers of the solution, the tip Ti is lowered vertically downward with the tube Tu slanted. Preferably, a method is employed. Specifically, when the tip Ti is inserted into the tube Tu as shown in FIG. 9 below, the tube Tu is tilted in advance, and the tip Ti is lowered in this state. , while avoiding the interface as much as possible by keeping the tip of the tip Ti along the wall surface of the tube Tu, the tip Ti is lowered to avoid the interface as much as possible, and the tip Ti penetrates into the lower layer. be able to.
As shown on the right side of Fig. 9, the tip of the tip Ti is fully inserted into the lower layer solution and the solution is sucked, thereby avoiding the suction of the interface and smoothly sucking the lower layer solution. can be recovered.

It should be noted that when the pipetting device 9 sucks and discharges the solution, the tube Tu can be carried out by the movable tube placing portion 21 of the cap removing/attaching device 13, which will be described later. The tube placing section 21 is configured so that the tube Tu can be tilted obliquely during the above-described pipetting.
In this way, it can be set so that the sample is pipetted at an angle so as not to aspirate the interface. In addition, a waste liquid bottle 51 is installed at substantially the center of FIG. 2 in order to dispose of the residual solution resulting from the pipetting operation.

In addition, when the pipetting device 9 sucks and collects the solution from the tip Ti as described above, a new tube Tu for discharging the collected solution is placed in a predetermined rack and the used tip Ti is discarded. It is preferable to prepare a tip disposal bin or the like.
Next, the first embodiment of the present invention further has the following configuration.

○ Cap removing/attaching device The cap removing/attaching device 13 in the first embodiment is a device for removing the cap C from the tube Tu or attaching the cap C by rotating the screw cap C as necessary. As shown in FIG. 10, in the first embodiment, the cap removing/attaching device 13 includes a movable tube placing portion 21, a substantially rectangular parallelepiped cap rotating/moving portion 15, and a disk-shaped cap placing portion. It has a portion 17 .

In the tube placing portion 21, the capped tube Tu to which the cap C is to be attached or removed is placed, and the lower portion of the tube Tu is supported so as to be in a non-rotatable state.
Next, in order to attach/detach the cap C, the cap rotating/moving unit 15 approaches the solution tube Tu with the cap carried to the tube placing unit 21, and the solution with the cap is placed under the solution tube Tu. It is arranged so that the tube Tu is located.

Specifically, as shown in FIG. 11 , holding portions 22 for the tubes Tu are formed in the tube mounting portion 21 , and the tubes Tu are inserted between the holding portions 22 .

The lower part and the bottom part of the holding part 22 are configured so that the lower part of the tube Tu can be supported, fixed and released, and when fixed, the rotation of the tube Tu is prevented. In this state, the cap rotating/moving unit 15 approaches from the top of the tube Tu, the cap lifting unit 16 descends, and the three cap clamping bars 18 descend to clamp the cap C, and then rotate. Cap C can be removed with .

Further, the removed screw-type cap C is placed on the disk-shaped placement portion 17 to complete the removal of the cap C. As shown in FIG.

On the other hand, when attaching the cap C, after placing the disk-shaped cap C on the upper part of the tube Tu with the cap C removed, the cap C is rotated and opened by the cap C. Close the part to complete the closure with cap C. While holding the lower part of the tube Tu, the cap rotating/moving part 15 approaches the upper part, the cap lifting part 16 descends, holds the cap C of the tube Tu, and rotates to lift the cap C. remove. Next, the cap C portion is placed on the upper portion of the placing portion 17 of the disk base, and the next step is performed.

The first embodiment of the present invention may further have the following configurations.

Movable stock unit In the first embodiment of the present invention, a slide-movable stock unit 23 is provided on which a plurality of sample tubes Tu with screw caps can be placed (Figs. 1 and 2). In the present invention, as shown in FIG. 12, it is provided in the area in front of the homogenizer device 25 .

Homogenizer Device The first embodiment of the present invention has an elevating homogenizer device 25 as shown in FIG. 13 which can be inserted into the sample tube Tu.
The homogenizer device 25 is configured to be vertically movable, and in front of the homogenizer device 25, tubes Tu stored in a tube rack, which is a movable stock unit 23 that can be positioned directly below the homogenizer device, move. , and brought to the lower part of the homogenizer device 25 .
In this state, the homogenizer device 25 descends vertically, passes through the hole of the slide cover 26 for preventing liquid dripping, and the tip of the homogenizer is inserted into the tube Tu into the liquid surface, and the homogenizer is operated. By doing so, it is possible to pulverize solids and the like.
After homogenization, the homogenizer device 25 is raised and left briefly on the tube Tu so that the liquid on the blades of the homogenizer drops into the tube Tu. Also, by moving the slide cover, dripping of liquid from the homogenizer device can be prevented. Further, the movable stock section (tube rack) 25 is moved and returned.

The homogenization in the embodiment of the present invention is performed in a row in the Y-axis direction for the 1st, 3rd, 5th, 7th, 9th, and 11th tubes Tu in order from the upper direction (back side of the paper surface) in the Y-axis direction as shown in FIG. and a row of homogenizers in the Y-axis direction for the 2nd, 4th, 6th, 8th, 10th, and 12th tubes Tu are arranged in parallel, and arranged so that homogenization is performed sequentially for these tubes Tu It is

○ Shaking device The first embodiment of the present invention has a shaking device 27 . In the shaking device 27, the sample tube Tu can be accommodated and shaken.

In the first embodiment of the present invention, a shaker 27 is provided as shown in FIG. Prior to shaking, a slide cover having a plurality of L-shaped portions slides in the longitudinal direction of the tube rack so that the cover portion is positioned above the tubes Tu. As a result, even if the rack 28 rotates, the tube Tu does not drop, and the rack 28 rotates left and right while the tube Tu is held by the rack 28 as shown in FIG. solution can be shaken to mix.

○Solvent Dispensing Unit The first embodiment of the present invention has a solvent (solution) dispensing device 29 as shown in FIG. The dispensing device 29 includes a solvent supply section 30 in which the screw-cap type sample tube placement section 21 of the cap removing/attaching device 15 moves and the solvent (solution) can be dispensed into the sample tube Tu. there is
For the solvent dispensing unit 29, a plurality of solvent supply units 30 are connected by thin flexible tubes to solvent stock bins arranged in a solvent stock unit 47 (Fig. 15).
The solvent supplied from the solvent stock bin of the stock section 47 is dispensed.
The reason why the sample tube mounting section 21 is movable from the cap removing/attaching device 15 to the solvent supply section 30 is that the solvent is often dispensed with the screw-type cap C removed. be.

That is, the sample tube Tu is moved with the cap C removed from the tube Tu by the cap removing/attaching device 13 described above, and the tube mounting part 21 moves, and the sample tube Tu with the cap C removed and the upper part open is opened. It is configured to be moved and positioned below the tip of the solvent supply section 30 of the solvent dispensing unit 29 .
The solvent dispensing unit 29 according to the first embodiment of the present invention shown in FIG. In order to dispense the solvent, the aforementioned tube mounting section 21 is moved to the lower part of the tip of one of the supply sections 30, and one of the supply sections 30 is slid in the direction of the arrow. , is positioned above the tube Tu, and is configured to be able to supply a solvent.

In addition, in order to keep the liquid volume in the tube Tu constant, it is possible to perform constant volume control using an optical sensor so that the shortage of the solution can be additionally supplied.
For example, when it is necessary to keep the volume at 30 ml, if the solution in the tube Tu is 28 ml, an optical sensor is used to supply an additional 2 ml so that the volume becomes 30 ml.

As for the solvent (solution) to be dispensed by the dispensing device 29, there is a stock unit 47 having a plurality of solvent stock bottles as shown in FIG. The solvent is sucked and discharged by the operation of the pump section 31-2.

The syringe pump unit 31-2 is a device for supplying a solvent such as a buffer stored in a predetermined solvent stock bottle into the tube Tu as needed.
The solvent stock bottle contains a solvent to be supplied, and is set to be sucked and discharged by the syringe pump 31-2.
Furthermore, the tube mounting part 21 in the solvent dispensing unit 29 described above is also a movable tube mounting part 21 when performing pipetting operations such as aspiration and discharge of the solution by the pipetting device 9 described above. As shown in FIG. 9, it is also configured to incline the tube Tu at a predetermined angle.

○Centrifugal Separator In the present invention, a centrifugal separator 35 for centrifuging the sample tube Tu is provided. By centrifuging, a residue and a supernatant can be separated in a homogenized solution. In addition, it is preferable to use a swing type rotor in the present invention.

In addition, in the centrifugal separation device 35, it is preferable that the aforementioned tube lifting device 7 vertically lowers the tube Tu when the sample tube Tu is inserted into the rotor. For this reason, it is preferable that the tube housing portion of the rotor is oriented vertically when the rotor stops rotating.
Furthermore, the first embodiment of the present invention has the following configuration.

(Fixed stock section) In the first embodiment of the present invention, a rack-type fixed stock section 33 on which the sample tubes Tu can be placed is provided. In addition, the stock section 33 also serves as a cooling unit so as to maintain a low temperature by a cooling trap 53 (FIG. 17).
A sample tube Tu can be appropriately placed on this stock section. Moreover, it can be usefully used when holding the sample tube Tu after the centrifugation operation and the relatively highly volatile organic solvent with the cap C removed.

○Treatment part by column In the first embodiment of the present invention, further purification can be performed by adding a purification step by column or filtration, which is preferable. A first embodiment of the present invention has a processing section 40 for processing by column as shown in FIG.
In the column processing section, a column mounting section 41 and a column solvent supply section 43 having a syringe or the like for supplying a buffer or the like from the top of the column are slid from the horizontal direction to be positioned above the column. .
A column pressure supply unit 44 is also provided for supplying pressure to the column by sending air or the like from the top of the attached column in order to promote the passage of the liquid through the column. A pressure supply syringe 31-1 for supplying air pressure to the pressure supply unit is shown in FIG.

O Centrifugal Evaporator Apparatus In the present invention, the centrifugal evaporator 49 is used to add drying and concentration steps, thereby enabling final adjustment of the sample. The first embodiment of the present invention shows a configuration including the centrifugal evaporator device 49 (FIG. 19).
The centrifugal evaporator device 49 is connected to a suction pump 55 installed on the right side of the automatic pretreatment device, accommodates a tube Tu with an open top, and centrifuges in a degassed state to volatilize the solvent. Allow to dry.
A predetermined solvent is supplied to the dried tube Tu to dissolve the contents, thereby finally completing the sample.

In the centrifugal evaporator device 49, when the sample tube Tu is inserted into the rotor, it is preferable that the tube elevating device 7 lowers the tube Tu vertically. , it is preferable to tilt the rotor or the centrifugal evaporator device 49 itself so that the tube Tu is vertically lowered and inserted into the tube receptacle to be housed in the rotor.

〇Others In addition to the tube rack section for placing new screw-type tubes Tu and pipetting operations, the waste liquid bottle 51, etc. for discarding the solution to be removed is not shown in Figure 2. can be provided. The screw-type tube Tu can also be placed on the fixed stock section 33 or the movable stock section 23 described above.

Next, an example of treatment of test samples in the automatic pretreatment apparatus according to the first embodiment of the present invention will be described. This disclosure is merely an example process and is not limited to this example process.

In particular, extraction, purification, and drying of veterinary drugs from food will be described with reference to the operating procedures shown in Tables 1 and 2. However, it goes without saying that each setting condition is not limited to those shown below.
In the following pretreatment method for analysis of veterinary drugs, pretreatment is performed in three steps of extraction→purification→concentration.
This flow is shown in Tables 1 and 2 for hydrophilic and lipophilic, respectively. These are processes for eluting veterinary drugs from samples such as agricultural products and foods into an extraction solvent.

本発明の第一の実施態様においては、以下のように自動前処理装置が稼働して、機器分析（LC-MS/MS）に供するサンプルを自動で調製することができる。
スクリュー式のチューブTuに対して、試料（食品、農作物等の粉砕しているものが好ましい）を添加する。当該試料の重量については記録しておく。

In the first embodiment of the present invention, the automatic pretreatment device operates as follows to automatically prepare a sample for instrumental analysis (LC-MS/MS).
A sample (preferably pulverized food, agricultural products, etc.) is added to the screw-type tube Tu. Record the weight of the sample.

Further, in the case of the first embodiment of the present invention, the screw cap C can be left attached to the tube Tu.

In this first embodiment, 12 samples can be processed. A sample can be loaded by arranging a tube Tu with a cap C containing a solid or liquid sample in the mobile stock section 23 shown in FIGS. In this state, automatic pretreatment by the automatic pretreatment apparatus of the first embodiment can be started.

1) When extracting a hydrophilic veterinary drug (1) Extraction process Moving and transferring the moving shaft 1 on the fixed rail 5 in the Y-axis direction with respect to the tube Tu with the cap C containing the sample Through the movement of the part 3 in the Z-axis direction on the movement axis 1, the tube lifting device 7 approaches the sample tube Ti with the cap C, and the sample cap clamping bar 18 descends from the upper part of the sample tube Ti. are used to hold the upper portion of the sample tube Tu.

Then, in this clamping state, the clamping bar 18 rises upward and continuously moves via the moving shaft 1 and the fixed rail 5, and the sample tube Tu containing the sample is placed on the tube of the cap removing/attaching device 13. move it to the storage area.

The clamping cap rotating part 15 of the cap removing/attaching device slides horizontally with respect to the sample tube Tu placed on the tube placing part 17, and rotates the cap part of the tube Tu to remove the cap C. The cap C is mounted on a disk-shaped cap mounting plate 17 . A sample-filled tube Tu placed on the tube placing portion of the detaching device is slid in the Y-axis direction and carried to a position for dispensing.

The supply unit 30 of one of the dispensing devices 29 is displaced in the X-axis direction with respect to the tube Tu moved to the position for dispensing, and is positioned above the tube Tu. Then, 15 ml of McIlpain buffer is added from the upper supply unit 30 . Next, the sample-filled tube Tu placed on the tube placing portion 21 of the detaching device is slid backward in the Y-axis direction and returned to its original position.
After adding the buffer, the tube Tu is clamped by the tube lifting device 7 from the tube placing portion 21 returned to its original position and placed on the transporting stock portion 23 in front of the homogenizer device 25 in the same manner as described above.
This operation is repeated, and the McIlvain buffer is added to the sample-containing tubes Tu sequentially placed on the mobile stock unit 23, and the McIlvain buffer is added to all the sample-containing tubes Tu. It is added and aligned with the mobile stock section 23 .

The movable stock unit 23 slides, and each tube Tu into which the buffer is added moves to the lower part of the homogenizer device 25 .

The homogenizer part 25 descends, the tip of the homogenizer 25 enters the liquid surface of the tube Tu, and homogenization is started. Homogenization is performed for 2 minutes, and after homogenization, the homogenization section 25 rises and the tip of the homogenizer separates from the sample tube Tu. Note that homogenization is performed in two rows depending on the number of samples.

The sample tube Tu after homogenization is transferred to the cap removing/attaching section 13 by the tube lifting device 7, and the cap C is attached in the reverse order of the previous steps. Then, the capped sample tube Tu is transferred to the centrifugal separator 35 by the tube elevating device 7 .

This operation is repeated an even number of times, and the sample tubes Tu after homogenization are placed in the centrifuge 35 so as to be symmetrical. If the number of samples to be processed is odd, it is preferable to insert an empty tube Tu to make the number even.

After the centrifugation, the tubes Tu are once sequentially transported to the cooling storage rack 33 and arranged. On the other hand, an empty tube Tu with its cap C opened is aligned with the rack 27 of the shaker. After centrifugation, the tubes Tu placed on the cooling rack are sequentially removed from the cap C by the cap removing device 13, and then sucked and discharged (pipetting part) in the tube placing section 21 to collect the supernatant. and poured into empty tubes Tu previously prepared in the rack 27 of the shaker.
That is, the sampled liquid is transferred to the tube Tu placed on the rack 27 of the shaker. In this embodiment, the aspirated supernatant liquid is transported by the suction/discharge mechanism of the pipetting device 9 attached to the transfer section and discharged to the new sample tube Tu.

McIl-Pain buffer is added to the residual sample tube Tu from which the buffer has been removed by the pipetting device 9 in the same manner as described above, transferred to the homogenizing device 25, and homogenized (for 1 minute). , followed by centrifugation.

After the homogenization and centrifugation, the supernatant produced by the second homogenization and centrifugation is aspirated into the tube Tu containing the supernatant previously and discharged by pipetting.

After two extraction operations, the tube Tu containing the collected buffer is placed at a position for dispensing, and McIlvain buffer is added to a constant volume so as to bring the volume up to 30 ml. An optical sensor is used so that the 30 ml line has a constant volume.

(2) Purification step These operations are repeated, and if the veterinary drug is present, a fraction containing the component is recovered. Next, 20 ml of water-saturated hexane for purification is added to the collected solution whose volume is fixed to 30 ml. The cap C is closed, and the tube Tu with the closed cap is stored in the rack 28 of the shaker 27 . After the storage is repeated a predetermined number of times, the rack 28 of the shaking device 27 is repeatedly rotated back and forth to perform shaking.

After the shaken sample tube Tu is centrifuged in the same manner as described above, the upper hexane layer is removed by the pipetting device 9 . The underlying McIlvain buffer layer is then recovered by pipetting.

At this time, as shown in FIG. 9, the sample tube Tu is tilted slightly in order to avoid suction at the interface between the hexane layer and the buffer layer during the pipetting operation. The chip Ti is operated such that the mounting portion of the chip Ti is lowered downward and the chip Ti is inserted into the buffer layer so as not to generate suction at the interface with the hexane layer.

While the purification operation with hexane is being performed, in the column processing unit 40, a buffer or the like is supplied from the top of the column to which the attached column is placed. The column solvent supply unit 43 slides horizontally to move to the top of the column, and supplies 5 ml of methanol from the attached supply syringe. In this way, the column layer is washed by passing the liquid through it at atmospheric pressure. Next, 5 ml of distilled water is passed through at atmospheric pressure.

Next, the pipetting device 9 approaches, a new tip Ti is attached, and 20 ml of the lower layer of the solution in the tube after removing the hexane layer is taken and supplied to the column (OASIS HLB). The column is pre-conditioned by passing 5 ml of methanol and 5 ml of distilled water under atmospheric pressure.

Next, 20 ml of the lower layer of the solution in the tube Tu was taken and applied to a column (OASIS HLB), passed under atmospheric pressure, and then air pressure was applied from above to completely pass through the column. Then 5 ml of distilled water is applied to wash the column. After passing the liquid by atmospheric pressure, air pressure is applied again from the upper part to pass the liquid further. A tube lifting device 7 removes the tube Tu containing the waste liquid that has passed through the column.
Then, a collection tube Tu is placed at the bottom of the column, 5 ml of ethanol is added as an eluent, pressure is applied, and the sample solution containing hydrophilic veterinary drugs is passed through the tube by atmospheric pressure. Collect in Tu. After that, the column is further passed under air pressure to elute the ethanol recovery liquid in the column.

(3) Concentration step The sample tube lifting device 7 approaches the open tube Tu containing the ethanol recovery liquid containing the hydrophilic veterinary drug, carries it to the centrifugal evaporator 37, and stores it in the rotor. . Place a blank of equal weight in the area of interest to balance the centrifuge.
A suction (vacuum) pump for decompression is activated to perform degassing while centrifugation is performed to volatilize the solvent and dry to dryness under reduced pressure.

Next, 1 ml of 50% methanol is added (0.5 ml in the case of dry product), the cap C is closed by the cap removing/attaching device 13, shaken by the shaking device 27 for 3 minutes, and stored at 4°C.

2) In the case of extracting lipophilic veterinary drugs (1) Extraction process A 50 ml tube Tu containing 1.5 g of a powder or paste sample is carried by the sample tube lifting device 7 to the cap removing device 13, and the cap C is removed. open. The unsealed tube Tu is transferred to the solution dispensing device 29, and acetonitrile is dispensed and added. The sample tube lifting device 7 transfers the dispensed tube Tu to the movable stock unit (slide rack) 23 under the homogenizer 25, and the movable stock unit (slide rack) 23 is slid under the homogenizer 25. Then the homogenizer 25 descends and is inserted into the liquid surface to crush and homogenize the solids.

After homogenization, the homogenizer device 25 is lifted and the slide rack 23 is slid back to its original position. The tubes Tu on the returned slide rack 23 are attached with caps C by the cap removing/attaching device 13, transferred to the centrifugal separator 35, and centrifuged for a predetermined time.

After completion of centrifugation, the tube is transferred to the tube placing portion 21 of the cap removing/attaching device 13 by the tube lifting device 7, and after the cap C is removed, the supernatant is aspirated by the pipetting device 9 equipped with the tip Ti. The supernatant is collected in a new tube Tu.
Next, the original tube Tu is moved to the solvent supply unit 30 of the solution dispensing device 29, and after acetonitrile is dispensed again, the tube Tu is transferred to the homogenization device 25 by the tube lifting device 7. It is transferred downward and homogenized in the same manner as described above.

After centrifugation is added after the homogenization, it is aspirated by pipetting with the pipetting device 9 as before, and the supernatant is added to the new tube Tu.
Through two extraction operations, the tube Tu containing the recovered buffer moves to a position for dispensing under the solvent dispensing unit, and McIlvain buffer is added to bring the volume up to 30 ml. be done. An optical sensor is used so that the 30 ml line has a constant volume.
After that, close the cap C and perform centrifugation, open the cap C, and collect the centrifugation supernatant in a new tube Tu. Repeat the above operations.

(2) Purification step These operations are repeated, and if the veterinary drug is present, a fraction containing the component is recovered. Next, 20 ml of acetonitrim-saturated hexane for purification is added to the recovered acetonitrile solution whose volume is 30 ml using the solvent dispensing unit 29 or the pipetting device 9 . The cap C is closed, and the tube Tu with the closed cap is stored in the tube rack 28 of the shaker 27 . After the storage is repeated a predetermined number of times, the tube rack 28 is shaken by repeatedly rotating back and forth.

After the shaken sample tube Tu is centrifuged in the same manner as described above, the upper hexane layer is removed by the pipetting device 9 .
After that, 15 ml of acetonitrile-saturated hexane is added to adjust the volume to 50 ml, and after the cap C is rotated by the cap removing/attaching device 13, the mixture is shaken for 10 minutes in the mixer section. Thereafter, centrifugal separation is performed at 3000 rpm for 3 minutes by a centrifuge, the cap C is removed by the cap removing device 13, and the upper hexane layer is removed.

After that, 10 ml of the lower layer is dispensed into a new tube Ti. At this time, in order to avoid suction at the interface between the hexane layer and the buffer layer during the pipetting operation, the sample tube Tu is tilted slightly, and in this tilted state, the tip Ti of the pipetting device is attached to the mounting part from above. descends downward and the tip Ti is inserted into the buffer layer so as not to cause suction at the interface with the hexane layer. 10ml of the lower acetonitrile layer is aliquoted and dispensed into a new tube Tu

(3) Concentration process The sample tube Tu elevating device approaches the open tube Tu containing the recovery liquid containing the oleaginous veterinary drug, carries it to the centrifugal evaporator 37, and stores it in the rotor. Place a blank of equal weight in the area of interest to balance the centrifuge.
The suction (vacuum) pump 55 for decompression is activated to perform degassing while centrifugation is performed to evaporate the solvent and dry under reduced pressure.
Add 0.5 ml of 50% methanol, close cap C, shake with a mixer for 3 minutes, and store at 4°C.

A specific example of treatment for veterinary drugs will be described below. However, it goes without saying that the present invention is not limited to these examples.
An example of the analysis of veterinary drugs is described below.
For the aforementioned purification steps (hydrophilic and lipophilic),
[Example 1] (Automatic pretreatment device sample: swine hydrophilicity)
As a sample, pig muscle was used, and the hydrophilic veterinary drug shown in Table 3 was added to the sample, and a recovery test was performed.

具体的には、豚の筋肉（生）（フードプロセッサーにより均一なペースト状にしたもの：500g）に表３に記載の４２種の親水性の動物用医薬品を添加し、添加・回収試験用の食品サンプルとした。

Specifically, the 42 types of hydrophilic veterinary drugs listed in Table 3 were added to pig muscle (raw) (made into a uniform paste by a food processor: 500 g), and the mixture was added and recovered for testing. A food sample.

Using the food sample, an extraction operation was performed using the automatic pretreatment apparatus of the present invention according to the procedure for hydrophilic samples described above.

The extracted sample was analyzed by LC/MS/MS under the following conditions.
<LC-MS/MS measurement conditions>
Column: Octadecylsilylated silica gel column Temperature: 40°C
Mobile phase: A solution (0.1% formic acid aqueous solution) and B solution (methanol containing 0.1% formic acid) are fed with a concentration gradient.
Mobile phase flow rate: 0.3 mL/min Ionization mode: ESI
Injection volume: 5 μL
As a result of the analysis, the number of veterinary drugs with a recovery rate of 70% or more and less than 120% was evaluated. Table 5 shows the results.

[Comparative Example 1] (Manual work sample: swine hydrophilicity)
Add 42 types of lipophilic veterinary drugs listed in Table 3 prepared in Example 1, and use food samples for addition/recovery tests, as in Example 1 as performed by the automatic pretreatment device Extraction of veterinary drugs was carried out manually by technicians following the procedure of . Other conditions are the same as those shown in Example 1.

[Example 2] (Automatic pretreatment device sample: hydrophilic shrimp)
Using shrimp as a sample, the hydrophilic veterinary drug shown in Table 3 was added to the sample, and a recovery test was performed.

Specifically, 42 types of lipophilic veterinary drugs listed in Table 3 were added to shrimp (made into a uniform paste by a food processor: 500 g) to prepare food samples for addition/recovery tests.

Using the food sample, an extraction operation was performed using the automatic pretreatment apparatus of the present invention according to the procedure for the lipophilic sample described above. Other conditions are the same as those shown in Example 1.

[Comparative Example 2] (Manual work sample: shrimp hydrophilic)
Using the shrimp prepared in Example 2, 42 types of lipophilic veterinary drugs listed in Table 3 were added, and the food samples for the addition/recovery test were used in Example 2. Extraction of veterinary drugs was manually carried out by technicians following the same procedure. Other conditions are the same as those shown in Example 2.

[Example 3] (Automatic pretreatment device sample: swine lipophilic animal drug)
As a sample, the pork muscle (raw) prepared in Example 1 was used, and the lipophilic veterinary drug shown in Table 4 was added to the sample, and a recovery test was performed.

具体的には、豚の筋肉（生）（フードプロセッサーにより均一なペースト状にしたもの：500g）に表４に記載の154種の親油性の動物医薬品を添加し、添加・回収試験用の食品サンプルとした。

Specifically, 154 types of lipophilic veterinary drugs listed in Table 4 were added to pig muscle (raw) (made into a uniform paste by a food processor: 500 g), and the food for the addition/recovery test was prepared. was taken as a sample.

Using the food sample, an extraction operation was performed using the automatic pretreatment apparatus of the present invention according to the procedure for the lipophilic sample described above.

The extracted sample was analyzed by LC/MS/MS under the following conditions.
<LC-MS/MS measurement conditions>
Column: Octadecylsilylated silica gel column Temperature: 40°C
Mobile phase: Liquid A (10% methanol containing 10 mmol/L ammonium acetate) and liquid B (methanol containing 10 mmol/L ammonium acetate) are fed with a concentration gradient.
Mobile phase flow rate: 0.3 mL/min Ionization mode: ESI
Injection volume: 5 μL
As a result of the analysis, the number of veterinary drugs with a recovery rate of 70% or more and less than 120% was evaluated. Table 5 shows the results.

[Comparative Example 3] (manual work sample: pig lipophilic)
Pork muscles (raw) prepared in Example 3, 154 types of lipophilic veterinary drugs listed in Table 4 were added, and food samples for addition/recovery tests were used in Example 3. Extraction of veterinary drugs was performed manually by technicians using procedures similar to those performed by .
Other conditions are the same as those shown in Example 3.

[Example 4] (Sample: shrimp lipophilic)
Using shrimp as a sample, the lipophilic veterinary drug shown in Table 4 was added to the sample, and a recovery test was performed.

Specifically, 154 types of lipophilic veterinary drugs listed in Table 4 were added to shrimp (made into a uniform paste by a food processor: 500 g) to prepare food samples for addition/recovery tests.

Using the food sample, an extraction operation was performed using the automatic pretreatment apparatus of the present invention according to the procedure for the lipophilic sample described above. Other conditions are the same as those shown in Example 3.

[Comparative Example 4] (Manual work sample: shrimp lipophilic)
Using the shrimp prepared in Example 4, 154 types of lipophilic veterinary drugs listed in Table 4 were added, and the food samples for the addition/recovery test were used in Example 4. Extraction of veterinary drugs was manually carried out by technicians following the same procedure. Other conditions are the same as those shown in Example 4.

結果として、手作業に比較して本自動前処理装置を利用すると、70％以上120％未満の回収率を得ることができる動物用医薬品の数が向上した。

As a result, the number of veterinary drugs for which a recovery rate of 70% or more and less than 120% can be obtained is increased using the automatic pretreatment device compared to manual processing.

Claims (4)

1) a transfer unit having a movement shaft and a transfer section that slides on the movement shaft and is movable within a predetermined plane;
2) a tube elevating device that is attached to the transfer unit and capable of clamping and releasing a screw-cap type sample tube to elevate it;
3) a vertically movable pipetting device that is attached to the transfer section, has a detachable predetermined tip portion, and sucks or discharges a liquid;
4) a screw cap-type sample tube mounting portion, and a cap removing/attaching device capable of holding and rotating the cap portion of the screw cap to attach/detach the cap, and placing the sample tube at a predetermined location;
5) a movable stock unit on which the screw-cap type sample tube is placed and which is slidable;
6) an elevating homogenizer device that can be inserted into the sample tube accommodated in the movable stock unit;
7) a rack for accommodating the sample tube, a shaking device having a shaking section capable of shaking the rack, and 8) a mounting section for the screw-cap type sample tube, and dispensing a solution into the sample tube. a solvent dispensing unit having a dispensing device with a pump section capable of
9) a centrifugal separator for centrifuging the sample tube;
An automatic pretreatment device for test samples,
When using the pipetting device to aspirate the lower layer of the two-layered solution in the sample tube, the tip attached to the pipetting device is lowered vertically while the sample tube is tilted. An automatic pretreatment device for test samples, configured to pass through the interface of a layered solution. The automatic pretreatment device further comprises
10) The automatic pretreatment apparatus for test samples according to claim 1, which includes a treatment section using a column. The automatic pretreatment device further comprises
11) The automatic pretreatment apparatus for test samples according to claim 2, comprising a centrifugal evaporator device. The automatic pretreatment device according to any one of claims 1 to 3, wherein the automatic pretreatment device for test samples is an automatic pretreatment device for test samples used for analysis of veterinary drugs.

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