CN115418115A - 一种吖啶橙制剂的制备方法 - Google Patents
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Abstract
本发明涉及一种吖啶橙制剂的制备方法，属于生物医药技术领域。制备方法包括配置含赋形剂和/或用于提高冻干过程中崩解温度的冻干保护剂的水溶液；将吖啶橙半氯化锌盐溶解于溶液中；将溶液分装入西林瓶中进行真空冷冻干燥，获得冻干粉；再配置吖啶橙冻干粉溶解液；将吖啶橙冻干粉溶解在吖啶橙冻干粉溶解液中，获得吖啶橙工作液。通过本发明提供的冷冻干燥工艺，可以制备有效期长达12个月的吖啶橙冻干粉，提高了试剂盒的有效期，提高了吖啶橙在生产、销售、使用中的便捷性。
Description
技术领域
本发明涉及一种吖啶橙制剂的制备方法，属于生物医药技术领域。
背景技术
现有技术中已有多种方法用于测试精子染色质和DNA的正常性。这些方法中通常都是通过应用与组蛋白结合(苯胺蓝)或与核酸结合(吖啶橙，色霉素)的染料，并应用组织学方法或流式细胞仪的方法来实现检测目的。较新的检测方法还包括DNA链断裂的检测，例如脱氧核糖核苷酸末端转移酶(TdT)介导的三磷酸脱氧核糖核苷酸(dUTP)缺口末端标记[短的用TUNEL(原位末端标记，ISEL)]，彗星试验或精子染色质扩散(SCD)试验。这些试验的结果之间互相关联，并且与精子形态学、精子活力和精子存活率相关。这些试验可以给出有关标准体外受精的受精率，还可能有关于自然妊娠率的额外信息。精子染色质结构试验(SCSA)能够预示体内和体外的受精成功率。
现有的用于进行精子染色质结构试验(SCSA)的试剂盒中的吖啶橙，工作浓度极低(约6mg/L)，见光易分解；直接配置低浓度吖啶橙工作液时，吖啶橙称量很少，误差较大。试剂盒中通常为100倍工作浓度的吖啶橙储备液，使用时再稀释至工作浓度。吖啶橙储备液在2～8℃避光的储存条件下，有效期为3个月左右。开封后制备为吖啶橙工作液的有效期约1-2周。现有技术中吖啶橙制剂的缺陷为吖啶橙储备液保存有效期较短，配套试剂盒的有效期较短，导致浪费严重。
发明内容
本发明的目的是为解决现有技术中吖啶橙储备液保存有效期短，配套试剂盒有效期短的技术问题。
为达到解决上述问题的目的，本发明所采取的技术方案是提供一种吖啶橙制剂的制备方法，包括以下步骤：
步骤1：配置含赋形剂和/或用于提高冻干过程中崩解温度的冻干保护剂的水溶液；
步骤2：将吖啶橙半氯化锌盐溶解于步骤1中获得的溶液中；
步骤3：将步骤2中的溶液分装入西林瓶中，将西林瓶放入真空冷冻干燥机进行真空冷冻干燥，获得冻干粉；
步骤4：配置吖啶橙冻干粉溶解液；
步骤5：将步骤3中获得的吖啶橙冻干粉溶解在步骤4的吖啶橙冻干粉溶解液中，获得吖啶橙工作液。
优选地，所述步骤1中的赋形剂包括蔗糖、乳糖脱脂、蛋白质及水解物、聚维酮、葡聚糖或山梨醇。
优选地，所述步骤1中的冻干保护剂包括甘露醇、甘氨酸、右旋糖苷、木糖醇，聚维酮。
优选地，所述步骤4中吖啶橙冻干粉溶解液中包括氯化钠、十二水磷酸氢二钠、二水乙二胺四乙酸二钠盐、一水柠檬酸和ProClin 300。
相比现有技术，本发明具有如下有益效果：
现有技术中吖啶橙储备液保存时间约3个月，保存时间短，容易造成浪费。同时过期的吖啶橙处理不简便，为使用带来困扰。通过本发明提供的冷冻干燥工艺，可以制备有效期长达12个月的吖啶橙冻干粉，提高了配套试剂盒的有效期，在生产、销售、使用方面带来更大的便捷性。
附图说明
图1为本发明一种吖啶橙制剂的制备方法工艺流程示意图。
具体实施方式
为使本发明更明显易懂，兹以优选实施例，并配合附图作详细说明如下：
如图1所示，本发明提供一种吖啶橙制剂的制备方法，包括以下步骤：
步骤1：配置含赋形剂和/或用于提高冻干过程中崩解温度的冻干保护剂的水溶液；
步骤2：将吖啶橙半氯化锌盐溶解于步骤1中获得的溶液中；
步骤3：将步骤2中的溶液分装入西林瓶中，将西林瓶放入真空冷冻干燥机进行真空冷冻干燥，获得冻干粉；
步骤4：配置吖啶橙冻干粉溶解液；
步骤5：将步骤3中获得的吖啶橙冻干粉溶解在步骤4的吖啶橙冻干粉溶解液中，获得吖啶橙工作液。
上述步骤1中的赋形剂包括蔗糖、乳糖脱脂、蛋白质及水解物、聚维酮、葡聚糖或山梨醇。
上述步骤1中的冻干保护剂包括甘露醇、甘氨酸、右旋糖苷、木糖醇，聚维酮。
上述步骤4中吖啶橙冻干粉溶解液中包括氯化钠、十二水磷酸氢二钠、二水乙二胺四乙酸二钠盐、一水柠檬酸和ProClin 300。
实施例
本发明提供一种吖啶橙的冷冻干燥工艺，
吖啶橙冷东干燥时浓度极小，干物质含量极少。为避免在冷东干燥时己经干燥的物质会被升华的气流带走，需要改善物质的浓度，需要增加干物质的含量，使冷冻后的产品能形成较理想的团块。
因此需要加入填充物质，使固体物质的浓度在4-25％之间。这些填充物或赋形剂是蔗糖、乳糖脱脂、蛋白质及水解物、聚维酮、葡聚糖、山梨醇等。
另外还需要加入一些物质可以提高冻干过程的崩解温度，以得到良好的产品并容易冻干。这些物质是甘露醇、甘氨酸、右旋糖苷、木糖醇，聚维酮等。
本发明提供：
1.一种吖啶橙的冷冻干燥工艺，其操作步骤为：
步骤1：称取蔗糖1.5g、甘氨酸1.5g，倒入50ml烧杯中,加入约30ml超纯水。搅拌至完全溶解。
步骤2：避免太阳光等强光直射。称取Acridine Orange吖啶橙半(氯化锌)盐0.03g，加入上述烧杯中，搅拌至完全溶解。
步骤3：将烧杯溶液使用玻璃棒转入50ml棕色容量瓶，用超纯水定容至50ml。再转入干燥洁净的棕色瓶。
步骤4：移液器量取75ul上述步骤3中溶液加入茶色西林瓶中，盖上内塞(下端1/2部分压入西林瓶，务必留孔，避免完全密闭)。
步骤5：在物料盘中加入3-5ml纯水，将西林瓶瓶底粘纯水后均匀分布摆放。每个物料盘可放置15-50个。
步骤6：将2个物料盘放入物料架，最上层留空。
步骤7：真空冷冻干燥机开启制冷0.5h后，此时确认冷肼温度低于-20℃。移开干燥架和玻璃罩，将载有茶色西林瓶的物料架放入冷肼，其上放置干燥架和玻璃罩，预冻1.5h。
步骤8：预冻1.5h后，此时确认冷肼温度低于-30℃。佩戴隔热手套，移开干燥架和玻璃罩，将物料架从冷肼中拿出，将干燥架放置在冷肼上，迅速转移载有西林瓶的物料盘至干燥架第二、三层(底层不放置)。物料盘中的水被冻成冰，可以固定西林瓶，同时冰块的逐渐融化可以提供转移过程和真空度降低之前的热量补偿，避免西林瓶中吖啶橙固体提前融化。盖上玻璃罩，关闭真空冷冻干燥机的放气阀门，开启真空泵，真空计。
步骤9：约1-2分钟后，真空计读数逐渐下降至100Pa以下，此时正式进入干燥阶段，物料盘中冰块只有少许融化，西林瓶中吖啶橙固体并未融化。开始计时4h。
步骤10：4h后，此时真空度应低于20Pa，西林瓶中吖啶橙固体中水分直接被升华，为蜂窝状冻干物。缓慢转动玻璃罩上方手柄，使玻璃罩中隔板下移，将内塞完全压进茶色西林瓶中，实现在接近真空状态中塞盖。
步骤11：将真空冷冻干燥机放气打开。等待1分钟后，真空度为正常大气压，关闭真空计，关闭真空泵，关闭制冷。取下玻璃罩，移走干燥架，取出物料盆。
步骤12：冻干效果评估：观察物料盘内金属表面应无液态水，棕色西林瓶内冻干粉应为黄色蜂窝状固体，无液态水。如果存在液态水，说明冻干过程失败。检查步骤8和步骤9，确认密闭性是否正常，真空计读数是否降低至100Pa以下，冻干时间是否达到4h。
步骤13：关闭真空冷冻干燥机电源。冷肼内冰霜自然融化，并排水。
步骤14：准备白色锁盖，将物料盘中的茶色西林瓶盖上锁盖。
步骤15：茶色西林瓶中为吖啶橙冻干粉，在2～8℃避光的储存条件下，自生产之日起有效期为12个月。
2.吖啶橙冻干粉溶解液的制备：
步骤1：称取氯化钠2.61g、十二水磷酸氢二钠13.53g、二水乙二胺四乙酸二钠盐0.12g、一水柠檬酸2.34g，倒入250ml烧杯中,加入约150ml超纯水。搅拌，至完全溶解。
步骤2：移液器量取ProClin 300 2.1ml，加入上述烧杯，搅拌，至完全溶解。
步骤3：将烧杯溶液转入250ml容量瓶，用超纯水定容至250ml。
3.吖啶橙冻干粉的溶解：
步骤1：取一只含有吖啶橙冻干粉的茶色西林瓶，去掉锁盖和内塞。
步骤2：量取吖啶橙冻干粉溶解液6.5ml，加入茶色西林瓶，盖上内塞，轻轻上下颠倒混匀。此为吖啶橙工作液，可直接用于精子染色质结构试验(SCSA)。溶解后2～8℃避光保存可用2周，使用EP管分装后，在-20℃冷冻避光保存可用12个月。
以上所述，仅为本发明的较佳实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员，在不脱离本发明的精神和范围的情况下，当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化，均为本发明的等效实施例；同时，凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变，均仍属于本发明的技术方案的范围内。
Claims (4)
1.一种吖啶橙制剂的制备方法，其特征在于，包括以下步骤：
步骤1：配置含赋形剂和/或用于提高冻干过程中崩解温度的冻干保护剂的水溶液；
步骤2：将吖啶橙半氯化锌盐溶解于步骤1中获得的溶液中；
步骤3：将步骤2中的溶液分装入西林瓶中，将西林瓶放入真空冷冻干燥机进行真空冷冻干燥，获得冻干粉；
步骤4：配置吖啶橙冻干粉溶解液；
步骤5：将步骤3中获得的吖啶橙冻干粉溶解在步骤4的吖啶橙冻干粉溶解液中，获得吖啶橙工作液。
2.根据权利要求1所述的一种吖啶橙制剂的制备方法，其特征在于：所述步骤1中的赋形剂包括蔗糖、乳糖脱脂、蛋白质及水解物、聚维酮、葡聚糖或山梨醇。
3.根据权利要求1所述的一种吖啶橙制剂的制备方法，其特征在于：所述步骤1中的冻干保护剂包括甘露醇、甘氨酸、右旋糖苷、木糖醇或聚维酮。
4.根据权利要求1所述的一种吖啶橙制剂的制备方法，其特征在于：所述步骤4中吖啶橙冻干粉溶解液中包括氯化钠、十二水磷酸氢二钠、二水乙二胺四乙酸二钠盐、一水柠檬酸和ProClin 300。
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| CN101129368A (zh) * | 2007-08-23 | 2008-02-27 | 姚俊华 | 含有兰索拉唑的冻干粉针 |
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| CN114235769A (zh) * | 2021-12-22 | 2022-03-25 | 深圳锐视生物科技有限公司 | 一种血细胞染色方法 |
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| CN101129368A (zh) * | 2007-08-23 | 2008-02-27 | 姚俊华 | 含有兰索拉唑的冻干粉针 |
| CN113652468A (zh) * | 2021-07-19 | 2021-11-16 | 江苏硕世生物科技股份有限公司 | 一种适用于核酸检测试剂盒冻干的冻干保护剂及其冻干方法 |
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